Compound containing a labile disulfide bond

ABSTRACT

A labile disulfide-containing compound under physiological conditions containing a labile disulfide bond and a transduction signal.

This application is a continuation-in-part of Ser. No. 09/3 12,351 filedon May 14, 1999, pending, which claims the benefit of U.S. Provisionalapplication No. 60/085,764, filed on May 16, 1998.

BACKGROUND

Bifunctional molecules, commonly referred to as crosslinkers, are usedto connect two molecules together. Bifunctional molecules can containhomo or heterobifunctionality. The disulfide linkage (RSSR′) may be usedwithin bifunctional molecules. The reversibility of disulfide bondformation makes them useful tools for the transient attachment of twomolecules. Disulfides have been used to attach a bioactive compound andanother compound (Thorpe, P. E. J. Natl. Cancer Inst. 1987, 79, 1101).The disulfide bond is reduced thereby releasing the bioactive compound.Disulfide bonds may also be used in the formation of polymers (Kishore,K., Ganesh, K. in Advances in Polymer Science, Vol. 21. Saegusa, T. Ed.,1993).

There are many commercially available reagents for the linkage of twomolecules by a disulfide bond. Additionally there are bifunctionalreagents that have a disulfide bond present. Typically, these reagentsare based on 3-mercaptopropionic acid, i.e. dithiobispropionate.However, the rate at which these bonds are broken under physiologicalconditions is slow. For example, the half life of a disulfide derivedfrom dithiobispropionimidate, an analog of 3-mercaptopropionic acid, is27 hours in vivo (Arpicco, S., Dosio, F., Brusa, P., Crosasso, P.,Cattel, L. Bioconjugate Chem. 1997,8. 327.). A stable disulfide bond isoften desirable, for example when purification of linked molecules orlong circulation in vivo is needed. For this reason, attempts have beenmade to make the disulfide less susceptible to cleavage.

It has been demonstrated that both stability, measured as reductionpotential, and rate, measured as rate constants, of disulfide reductionare both related to the acidity of the thiols which constitute thedisulfide. Additional factors that may affect the rate of reduction aresteric interactions, and intramolecular disulfide cleavage. Looking atthe difference in the rates for the reactions RSH+R′SSR′→RSSR′+R′SH andRSH+R″SSR″→RSSR″+R″SH, it has been demonstrated that log k″/k′=β(pK_(a)^(R′)−pK_(a) ^(R″)), where k′ and k″ are the rate constant for thereactions with R′SSR′ and R″SSR″ respectively, pK_(a) ^(R′) and pK_(a)^(R″) are the acidities of the thiol groups R′SH and R″SH, and β is aconstant determined empirically to be 0.72. From this equation, onewould predict that the reduction of a disulfide composed from relativelyacidic thiols would be reduced more quickly than one composed of lessacidic thiols. In support of this observation, it has been demonstratedthat the disulfides cystine (pK_(a) 8.3) and cystamine (pK_(a) 8.2) arereduced 3–15 times faster than oxidized glutathione (pK_(a) 8.9) (Bulaj,G., Kortemme, T., Goldenberg, D. P. Biochemistry 1998, 37, 8965).

It has been demonstrated that both stability (thermodynamics), measuredas reduction potential (Keire D. A. J. Org. Chem. 1992, 57, 123), andrate (kinetics), measured as rate constants, of disulfide reduction areboth related to the acidity of the thiols which constitute the disulfide(Szajewski, R. P., Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 2011).The increase in acidity of a thiol is dependent upon one or more of thefollowing structural factors: the presence of electron withdrawinggroups which stabilize the thiolate through sigma and pi bonds(inductive effect), the presence of electron withdrawing groups thatstabilize the thiolate through space or solvent (field effects), pibonds which allow the negative charge to be placed on other atoms(resonance stabilization), and hydrogen bond donating groups within themolecule that can interact internally with the thiolate. For example,cysteine has an amino group two atoms from the thiol, which is moreelectron withdrawing than the amide nitrogen that is two atoms from thethiol in glutathione. As a consequence of this difference in electronwithdrawing groups, the thiol of cysteine is 0.6 pK units more acidicthan glutathione, and as mentioned previously, cystine is reduced 3–15times faster than oxidized glutathione. Another example of a relativelyacidic thiol is 5-thio-2-nitrobenzoic acid, pK_(a) 5. Its acidity is dueto resonance stabilization and inductive effects. Its disulfide israpidly reduced by all standard alkyl thiols and its colored thiolatemakes it a convenient assay for thiol concentration.

SUMMARY

Described in a preferred embodiment is a process for the delivery of acompound to a cell, comprising associating a compound, containing adisulfide bond that can be cleaved under physiological conditions, witha polymer, then delivering the polymer to the cell. The polymer maycomprise a first polymer and a second polymer. The first polymer and thesecond polymer may comprise nucleic acids, proteins, genes, antisensepolymers, DNA/RNA hybrids, or synthetic polymers.

In another preferred embodiment, a biologically active compound isassociated with a disulfide-containing compound, comprising: thedisulfide-containing compound having a labile disulfide bond that isselected from the group consisting of (a) a disulfide bond that iscleaved more rapidly than oxidized glutathione and (b) a disulfide bondconstructed from thiols in which one of the constituent thiols has alower pKa than glutathione and (c) a disulfide bond that is activated byintramolecular attack from a free thiol.

In another preferred embodiment, a compound is provided for insertinginto an organism, comprising: the compound having a disulfide bond thatis labile under physiologic conditions selected from the groupconsisting of (a) a disulfide bond that is cleaved more rapidly thanoxidized glutathione and (b) a disulfide bond constructed from thiols inwhich one of the constituent thiols has a lower pKa than glutathione and(c) a disulfide bond that is activated by intramolecular attack from afree thiol.

In another preferred embodiment, a process is provided for forming acompound having a labile disulfide bond for use with an organism,comprising: forming the compound having a disulfide bond selected fromthe group consisting of (i) a disulfide bond that is cleaved morerapidly than oxidized glutathione, and (ii) a disulfide bond constructedfrom thiols in which one of the constituent thiols has a lower pKa thanglutathione, and (iii) a disulfide bond that is activated byintramolecular attack from a free thiol; inserting the compound into theorganism.

In another preferred embodiment, a process is described for compacting anucleic acid for delivery to a cell, comprising associating a polymercontaining a disulfide bond with a nucleic acid and delivering thenucleic acid to the cell.

In another preferred embodiment, a process is described for compacting anucleic acid for delivery to a cell comprising associating a polymerwith the nucleic acid, then associating a compound containing adisulfide bond that can be cleaved under physiological conditions withthe nucleic acid polymer complex, then delivering the complex to a cell.

In another preferred embodiment, a process is described for compacting anucleic acid for delivery to a cell, comprising associating a polymercontaining a disulfide bond with a nucleic acid, then associatinganother polymer with the disulfide containing polymer—nucleic acidcomplex, then delivering the complex to the cell.

In another preferred embodiment, a process is described for compacting anucleic acid for delivery to a cell comprising associating a polymerwith the nucleic acid, then associating a compound containing adisulfide bond that can be cleaved under physiological conditions withthe nucleic acid polymer complex, then associating another polymer withthe complex, then delivering the complex to a cell.

In another preferred embodiment, a compound is described which containsa disulfide bond that can be cleaved under physiological conditions andpossesses heterobifunctional or homobifunctional groups. Such a compoundcan be described as a disulfide containing bifunctional molecule.A₁-S—S-A₂

More particularly, a compound that contains an aliphatic disulfide bondwith one or more electronegative (electron withdrawing groups)substituted alpha or beta to one or both of the sulfur atoms. Thesegroups serve to lower the pK_(a) of the constituent thiols.

Where R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈— at least one of which is anelectronegative atom or functionality such as OH, OR (an ether),NH₂,(also secondary, tertiary, and quaternary amines), SO₃ ⁻, COOH, COOR(an ester), CONH₂, CONR₂ (substituted amide), a halogen (F, Cl, Br, I),NO₂. L is defined as a linker or spacer group that provides a connectionbetween the disulfide and the reactive heterobifunctional orhomobifunctional groups, A₁ and A₂. L may or may not be present and maybe chosen from a group that includes alkanes, alkenes, alkynes, esters,ethers, glycerol, amide, urea, saccharides, polysaccharides, heteroatomssuch as oxygen, sulfur, or nitrogen. The spacer may be charge positive,charge negative, charge neutral, or zwitterionic. A₁ and A₂ are reactivegroups they may be identical as in a homobifunctional bifunctionalmolecule, or different as in a heterobifunctional bifunctional molecule.In a preferred embodiment, the disulfide compounds contain reactivegroups that can undergo acylation or alkylation reactions. Such reactivegroups include (but not limited to) isothiocyanate, isocyanate, acylazide, acid halide, O-acyl urea, N-hydroxysuccinimide esters,succinimide esters, amide, urea, sulfonyl chloride, aldehyde, ketone,ether, epoxide, carbonate, alkyl halide, imidoester, carboxylate,alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene) oranhydrides.

If functional group A1,A2 is an amine then A1,A2 can react with (but notrestricted to) an activated carboxylic acid, isothiocyanate, isocyanate,acyl azide, alkyl halide, acid halide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,amide, carboxylate, or alkylphosphate, arylhalides(difluoro-dinitrobenzene) or anhydrides. In other terms when functionA1,A2 is an amine, then an acylating or alkylating agent can react withthe amine.

If functional group A1,A2 is a sulflhydryl then A1,A2 can react with(but not restricted to) a haloacetyl derivative, activated carboxylicacid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

If functional group A1,A2 is carboxylate then A1,A2 can react with (butnot restricted to) a diazoacetate, alcohol, thiol or an amine once theacid has been activated.

If functional group A1,A2 is an hydroxyl then A1,A2 can react with (butnot restricted to) an activated carboxylic acid, epoxide, oxirane, or anamine in which carbonyldiimidazole is used.

If functional group A1,A2 is an aldehyde or ketone then A1,A2 can reactwith (but not restricted to) an hydrazine, hydrazide derivative, amine(to form a Schiff Base that may or may not be subsequently reduced byreducing agents such as NaCNBH₃), or a diol to form an acetal or ketal.

If functional group A1,A2 is activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, N-hydroxysuccinimide ester, sulfonyl chloride,aldehyde, ketone, epoxide, carbonate, imidoester, alkylphosphate,arylhalides (difluoro-dinitrobenzene), anhydride, alkyl halide, or acidhalide, p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenylester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyridinium,or carbonyl dimethylaminopyridinium, then A1,A2 can react with (but notrestricted to) an amine, a hydroxyl, hydrazine, hydrazide, or sulfhydrylgroup.

If functional group A1,A2 an activated carboxylic acid, haloacetylderivative, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) then A1,A2 canreact with (but not restricted to) a sulfhydryl.

If functional group A1,A2 is an aldehyde, ketone, epoxide, oxirane, oran amine in which carbonyldiimidazole or N,N′-disuccinimidyl carbonateis used, then A1,A2 can react with (but not restricted to) a hydroxyl.

If functional group A1,A2 is a hydrazine, hydrazide derivative, or amine(primary or secondary) then A1,A2 can react with (but not restricted to)an aldehyde or ketone (to form a Schiff Base that may or may not bereduced by reducing agents such as NaCNBH₃).

Additionally, a compound which contains an aromatic disulfide bond inwhich the sulfur atom is bonded directly to the aromatic ring. The ringmay contain 5 or more atoms.

R₁–R₄, R₆–R₉— The substitution pattern on the ring may be varied toalter the reduction potential of the disulfide bond. The substiuents maybe selected from the group that includes but is not limited to OH, OR(an ether), NH₂,(also secondary, tertiary, and quaternary amines), SO₃⁻, COOH, COOR (an ester), CONH₂, CONR₂ (substituted amide), a halogen(F, Cl, Br, I), NO₂, CH₃ (or longer branched or straight chain,saturated, or unsaturated aliphatic group). L is defined as a linker orspacer group that provides a connection between the disulfide and thereactive heterobifinctional or homobifunctional groups. L may or may notbe present and may be chosen from a group that includes alkanes,alkenes, esters, ethers, glycerol, amide, saccharides, polysaccharides,heteroatoms such as oxygen, sulfur, or nitrogen. The spacer may becharge positive, charge negative, charge neutral, or zwitterionic. R₅,R₁₀—are reactive groups they may be identical as in a homobifunctionalbifunctional molecule, or different as in a heterobifunctionalbifunctional molecule. In a preferred embodiment, the disulfidecompounds contain reactive groups that can undergo acylation oralkylation reactions. Such reactive groups include isothiocynanate,isocynanate, acyl azide, N-hydroxysuccinimide esters, succinimideesters, sulfonyl chloride, aldehyde, epoxide, carbonate, imidoester,carboxylate, alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene)or succinic anhydride.

If functional group R5, R10 is an amine then R5, R10 can react with (butnot restricted to) an activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, alkyl halide, acid halide, N-hydroxysuccinimideester, sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,imidoester, amide, carboxylate, or alkylphosphate, arylhalides(difluoro-dinitrobenzene) or anhydrides. In other terms when functionR5, R10 is an amine, then an acylating or alkylating agent can reactwith the amine.

If functional group R5, R10 is a sulfhydryl then R5, R10 can react with(but not restricted to) a haloacetyl derivative, activated carboxylicacid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

If functional group R5, R10 is carboxylate then R5, R10 can react with(but not restricted to) a diazoacetate, alcohol, thiol or an amine oncethe acid has been activated.

If functional group R5, R10 is an hydroxyl then R5, R10 can react with(but not restricted to) an activated carboxylic acid, epoxide, oxirane,or an amine in which carbonyldiimidazole is used.

If functional group R5, R10 is an aldehyde or ketone then R5, R10 canreact with (but not restricted to) an hydrazine, hydrazide derivative,amine (to form a Schiff Base that may or may not be subsequently reducedby reducing agents such as NaCNBH₃), or a diol to form an acetal orketal.

If functional group R5, R10 is activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydride, alkylhalide, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium, then R5, R10can react with (but not restricted to) an amine, a hydroxyl, hydrazine,hydrazide, or sulfhydryl group.

If functional group R5, R10 an activated carboxylic acid, haloacetylderivative, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) then R5, R10can react with (but not restricted to) a sulfhydryl. If functional groupR5, R10 is an aldehyde, ketone, epoxide, oxirane, or an amine in whichcarbonyldiimidazole or N,N′-disuccinimidyl carbonate is used, then R5,R10 can react with (but not restricted to) a hydroxyl.

If functional group R5, R10 is a hydrazine, hydrazide derivative, oramine (primary or secondary) then R5, R10 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

Additionally, a compound which contains a disulfide bond that isconnected directly to a heterocyclic ring. The heterocyclic ring may bearomatic or aliphatic. The heterocyclic ring may contain 5 or more atomsof which 1 or more is a heteroatom (O, N, S, P), and the rest beingcarbon atoms

H is a heteroatom selected from the group including sulfur, oxygen,nitrogen, or phosphorus. R₁–R₃, R₅–R₇ are substiuents that may beselected from the group that includes but is not limited to OH, OR (anether), NH₂,(also secondary, tertiary, and quaternary amines), SO₃ ⁻,COOH, COOR (an ester), CONH₂, CONR₂ (substituted amide), a halogen (F,Cl, Br, I), NO₂, CH₃ (or longer branched or straight chain, saturated,or unsaturated aliphatic group). The substitution pattern on thearomatic ring may be varied to alter the reduction potential of thedisulfide bond. L is defined as a linker or spacer group that provides aconnection between the disulfide and the reactive heterobifunctional orhomobifunctional groups. L may or may not be present and may be chosenfrom a group that includes alkanes, alkenes, esters, ethers, glycerol,amide, saccharides, polysaccharides, heteroatoms such as oxygen, sulfur,or nitrogen. The spacer may be charge positive, charge negative, chargeneutral, or zwitterionic. R₄, R₈ are reactive groups they may beidentical as in a homobifunctional bifunctional molecule, or differentas in a heterobifunctional bifunctional molecule. In a preferredembodiment, the disulfide compounds contain reactive groups that canundergo acylation or alkylation reactions. Such reactive groups includeisothiocynanate, isocynanate, acyl azide, N-hydroxysuccinimide esters,succinimide esters, sulfonyl chloride, aldehyde, epoxide, carbonate,imidoester, carboxylate, alkylphosphate, arylhalides (e.g.difluoro-dinitrobenzene) or succinic anhydride.

If functional group R4, R8 is an amine then R4, R8 can react with (butnot restricted to) an activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, alkyl halide, acid halide, N-hydroxysuccinimideester, sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,imidoester, amide, carboxylate, or alkylphosphate, arylhalides(difluoro-dinitrobenzene) or anhydrides. In other terms when functionR4, R8 is an amine, then an acylating or alkylating agent can react withthe amine.

If functional group R4, R8 is a sulfhydryl then R4, R8 can react with(but not restricted to) a haloacetyl derivative, activated carboxylicacid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

If functional group R4, R8 is carboxylate then R4, R8 can react with(but not restricted to) a diazoacetate, alcohol, thiol or an amine oncethe acid has been activated.

If functional group R4, R8 is an hydroxyl then R4, R8 can react with(but not restricted to) an activated carboxylic acid, epoxide, oxirane,or an amine in which carbonyldiimidazole is used.

If functional group R4, R8 is an aldehyde or ketone then R4, R8 canreact with (but not restricted to) an hydrazine, hydrazide derivative,amine (to form a Schiff Base that may or may not be subsequently reducedby reducing agents such as NaCNBH₃), or a diol to form an acetal orketal.

If functional group R4, R8 is activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, N-hydroxysuccinimide ester, sulfonyl chloride,aldehyde, ketone, epoxide, carbonate, imidoester, alkylphosphate,arylhalides (difluoro-dinitrobenzene), anhydride, alkyl halide, or acidhalide, p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenylester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyridinium,or carbonyl dimethylaminopyridinium, then R4, R8 can react with (but notrestricted to) an amine, a hydroxyl, hydrazine, hydrazide, or sulfhydrylgroup.

If functional group R4, R8 an activated carboxylic acid, haloacetylderivative, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) then R4, R8can react with (but not restricted to) a sulfhydryl.

If functional group R4, R8 is an aldehyde, ketone, epoxide, oxirane, oran amine in which carbonyldiimidazole or N, N′-disuccinimidyl carbonateis used, then R4, R8 can react with (but not restricted to) a hydroxyl.

If functional group R4, R8 is a hydrazine, hydrazide derivative, oramine (primary or secondary) then R4, R8 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

Additionally, a compound which contains a disulfide bond that isconnected directly to a ring system(aromatic or non-aromatic) throughone of the sulfur atoms and to a aliphatic carbon through the othersulfur atom. The cyclic ring may contain 5 or more atoms.

R₁–R₄ are substiuents selected from the group that includes but is notlimited to H, OH, OR (an ether), NH₂,(also secondary, tertiary, andquaternary amines), SO₃ ⁻, COOH, COOR (an ester), CONH₂, CONR₂(substituted amide), a halogen (F, Cl, Br, I), NO₂, CH₃ (or longerbranched or straight chain, saturated, or unsaturated aliphatic group).The substitution pattern on the aromatic ring may be varied to alter thereduction potential of the disulfide bond. R₆–R₉ are substiuentsselected from the group that includes but is not limited to H, OH, OR(an ether), NH₂,(also secondary, tertiary, and quaternary amines), SO₃^(b−), COOH, COOR (an ester), CONH₂, CONR₂ (substituted amide), ahalogen (F, Cl, Br, I), NO₂, CH₃ (or longer branched or straight chain,saturated, or unsaturated aliphatic group). L is defined as a linker orspacer group that provides a connection between the disulfide and thereactive heterobifunctional or homobifunctional groups. L may or may notbe present and may be chosen from a group that includes alkanes,alkenes, esters, ethers, glycerol, amide, saccharides, polysaccharides,heteroatoms such as oxygen, sulfur, or nitrogen. The spacer may becharge positive, charge negative, charge neutral, or zwitterionic. R₅,and R₁₀ are reactive groups that may be identical as in ahomobifunctional bifunctional molecule, or different as in aheterobifunctional bifunctional molecule. In a preferred embodiment, thedisulfide compounds contain reactive groups that can undergo acylationor alkylation reactions. Such reactive groups include isothiocynanate,isocynanate, acyl azide, N-hydroxysuccinimide esters, succinimideesters, sulfonyl chloride, aldehyde, epoxide, carbonate, imidoester,carboxylate, alkylphosphate, arylhalides (e.g. difluoro-dinitrobenzene)or succinic anhydride.

If functional group R5, R10 is an amine then R5, R10 can react with (butnot restricted to) an activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, alkyl halide, acid halide, N-hydroxysuccinimideester, sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,imidoester, amide, carboxylate, or alkylphosphate, arylhalides(difluoro-dinitrobenzene) or anhydrides. In other terms when functionR5, R10 is an amine, then an acylating or alkylating agent can reactwith the amine.

If functional group R5, R10 is a sulfhydryl then R5, R10 can react with(but not restricted to) a haloacetyl derivative, activated carboxylicacid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

If functional group R5, R10 is carboxylate then R5, R10 can react with(but not restricted to) a diazoacetate, alcohol, thiol or an amine oncethe acid has been activated.

If functional group R5, R10 is an hydroxyl then R5, R10 can react with(but not restricted to) an activated carboxylic acid, epoxide, oxirane,or an amine in which carbonyldiimidazole is used.

If functional group R5, R10 is an aldehyde or ketone then R5, R10 canreact with (but not restricted to) an hydrazine, hydrazide derivative,amine (to form a Schiff Base that may or may not be subsequently reducedby reducing agents such as NaCNBH₃), or a diol to form an acetal orketal.

If functional group R5, R10 is activated carboxylic acid,isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,alkylphosphate, arylhalides (difluoro-dinitrobenzene), anhydride, alkylhalide, or acid halide, p-nitrophenyl ester, o-nitrophenyl ester,pentachlorophenyl ester, pentafluorophenyl ester, carbonyl imidazole,carbonyl pyridinium, or carbonyl dimethylaminopyridinium, then R5, R10can react with (but not restricted to) an amine, a hydroxyl, hydrazine,hydrazide, or sulfhydryl group.

If functional group R5, R10 an activated carboxylic acid, haloacetylderivative, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) then R5, R10can react with (but not restricted to) a sulfhydryl.

If functional group R5, R10 is an aldehyde, ketone, epoxide, oxirane, oran amine in which carbonyldiimidazole or N,N′-disuccinimidyl carbonateis used, then R5, R10 can react with (but not restricted to) a hydroxyl.

If functional group R5, R10 is a hydrazine, hydrazide derivative, oramine (primary or secondary) then R5, R10 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

Additionally, a compound which contains a disulfide bond that isconnected directly to a heterocyclic ring system through one of thesulfur atoms and to a aliphatic carbon through the other sulfur atom.The heterocyclic ring may contain 5 or more atoms of which 1 or more isa heteroatom (O, N, S, P) or combinations of heteroatoms, and the rest

being carbon atoms.

H is a heteroatom selected from the group including sulfur, oxygen,nitrogen, or phosphorus. Where R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,R₉, R10,R11, R12, R14- at least one of which is an electronegative atom orfunctionality such as OH, OR (an ether), NH₂, (also secondary, tertiary,and quaternary amines), SO₃ ⁻, COOH, COOR (an ester), CONH₂, CONR₂(substituted amide), a halogen (F, Cl, Br, I), NO₂. L is defined as alinker or spacer group that provides a connection between the disulfideand the reactive heterobifunctional or homobifunctional groups, A₁ andR9. L may or may not be present and may be chosen from a group thatincludes alkanes, alkenes, alkynes, esters, ethers, glycerol, amide,urea, saccharides, polysaccharides, heteroatoms such as oxygen, sulfur,or nitrogen. The spacer may be charge positive, charge negative, chargeneutral, or zwitterionic. A₁ and R9 are reactive groups they may beidentical as in a homobifunctional bifunctional molecule, or differentas in a heterobifunctional bifunctional molecule. In a preferredembodiment, the disulfide compounds contain reactive groups that canundergo acylation or alkylation reactions. Such reactive groups include(but not limited to) isothiocynanate, isocynanate, acyl azide, acidhalide, O-acyl urea, N-hydroxysuccinimide esters, succinimide esters,amide, urea, sulfonyl chloride, aldehyde, ketone, ether, epoxide,carbonate, alkyl halide, imidoester, carboxylate, alkylphosphate,arylhalides (e.g. difluoro-dinitrobenzene) or anhydrides.

If functional group A1,R9 is an amine then A1,R9 can react with (but notrestricted to) an activated carboxylic acid, isothiocyanate, isocyanate,acyl azide, alkyl halide, acid halide, N-hydroxysuccinimide ester,sulfonyl chloride, aldehyde, ketone, epoxide, carbonate, imidoester,amide, carboxylate, or alkylphosphate, arylhalides(difluoro-dinitrobenzene) or anhydrides. In other terms when functionA1,R9 is an amine, then an acylating or alkylating agent can react withthe amine.

If functional group A1,R9 is a sulfhydryl then A1,R9 can react with (butnot restricted to) a haloacetyl derivative, activated carboxylic acid,maleimide, aziridine derivative, acryloyl derivative, fluorobenzenederivatives, or disulfide derivative (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid{TNB} derivatives).

If functional group A1,R9 is carboxylate then A1,R9 can react with (butnot restricted to) a diazoacetate, alcohol, thiol or an amine once theacid has been activated.

If functional group A1,R9 is an hydroxyl then A1,R9 can react with (butnot restricted to) an activated carboxylic acid, epoxide, oxirane, or anamine in which carbonyldiimidazole is used.

If functional group A1,R9 is an aldehyde or ketone then A1,R9 can reactwith (but not restricted to) an hydrazine, hydrazide derivative, amine(to form a Schiff Base that may or may not be subsequently reduced byreducing agents such as NaCNBH₃), or a diol to form an acetal or ketal.

If functional group A1,R9 is activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, N-hydroxysuccinimide ester, sulfonyl chloride,aldehyde, ketone, epoxide, carbonate, imidoester, alkylphosphate,arylhalides (difluoro-dinitrobenzene), anhydride, alkyl halide, or acidhalide, p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenylester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyridinium,or carbonyl dimethylaminopyridinium, then A1,R9 can react with (but notrestricted to) an amine, a hydroxyl, hydrazine, hydrazide, or sulfhydrylgroup.

If functional group A1,R9 an activated carboxylic acid, haloacetylderivative, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives) then A1,R9 canreact with (but not restricted to) a sulfhydryl.

If functional group A1,R9 is an aldehyde, ketone, epoxide, oxirane, oran amine in which carbonyldiimidazole or N,N′-disuccinimidyl carbonateis used, then A1,R9 can react with (but not restricted to) a hydroxyl.

If functional group A1,R9 is a hydrazine, hydrazide derivative, or amine(primary or secondary) then A1,R9 can react with (but not restricted to)an aldehyde or ketone (to form a Schiff Base that may or may not bereduced by reducing agents such as NaCNBH₃).

Additionally, a compound which contains a disulfide bond that isconnected directly to a heterocyclic ring system (aromatic ornon-aromatic) through one of the sulfur atoms and to an aromatic ringsystem through the other sulfur atom. The heterocyclic ring may contain5 or more atoms of which 1 or more is a heteroatom (O, N, S, P) orcombinations of heteroatoms, and the rest being carbon atoms.

H is a heteroatom selected from the group including sulfur, oxygen,nitrogen, or phosphorus. Where R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₁₀, R₁₁,R₁₂, R₁₃— at least one of which is an electronegative atom orfunctionality such as OH, OR (an ether), NH₂, (also secondary, tertiary,and quaternary amines), SO₃ ⁻, COOH, COOR (an ester), CONH₂, CONR₂(substituted amide), a halogen (F, Cl, Br, I), NO₂. L is defined as alinker or spacer group that provides a connection between the disulfideand the reactive heterobifunctional or homobifunctional groups, R₉ andR₁₄. L may or may not be present and may be chosen from a group thatincludes alkanes, alkenes, alkynes, esters, ethers, glycerol, amide,urea, saccharides, polysaccharides, heteroatoms such as oxygen, sulfur,or nitrogen. The spacer may be charge positive, charge negative, chargeneutral, or zwitterionic. R₉ and R₁₄ are reactive groups they may beidentical as in a homobifunctional bifunctional molecule, or differentas in a heterobifunctional bifunctional molecule. In a preferredembodiment, the disulfide compounds contain reactive groups that canundergo acylation or alkylation reactions. Such reactive groups include(but not limited to) isothiocynanate, isocynanate, acyl azide, acidhalide, O-acyl urea, N-hydroxysuccinimide esters, succinimide esters,amide, urea, sulfonyl chloride, aldehyde, ketone, ether, epoxide,carbonate, alkyl halide, imidoester, carboxylate, alkylphosphate,arylhalides (e.g. difluoro-dinitrobenzene) or anhydrides.

If functional group R9, R14 is an amine then R9, R14 can react with (butnot restricted to) an activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, alkyl halide, acid halide, N-hydroxysuccinimideester, sulfonyl chloride, aldehyde, ketone, epoxide, carbonate,imidoester, amide, carboxylate, or alkylphosphate, arylhalides(difluoro-dinitrobenzene) or anhydrides. In other terms when functionR9,R14 is an amine, then an acylating or alkylating agent can react withthe amine.

If functional group R9,R14 is a sulfhydryl then R9,R14 can react with(but not restricted to) a haloacetyl derivative, activated carboxylicacid, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid{TNB} derivatives).

If functional group R9,R14 is carboxylate then R9,R1 4 can react with(but not restricted to) a diazoacetate, alcohol, thiol or an amine oncethe acid has been activated.

If functional group R9,R14 is an hydroxyl then R9,R14 can react with(but not restricted to) an activated carboxylic acid, epoxide, oxirane,or an amine in which carbonyldiimidazole is used.

If functional group R9,R14 is an aldehyde or ketone then R9,R14 canreact with (but not restricted to) an hydrazine, hydrazide derivative,amine (to form a Schiff Base that may or may not be subsequently reducedby reducing agents such as NaCNBH₃), or a diol to form an acetal orketal.

If functional group R9,R14 is activated carboxylic acid, isothiocyanate,isocyanate, acyl azide, N-hydroxysuccinimide ester, sulfonyl chloride,aldehyde, ketone, epoxide, carbonate, imidoester, alkylphosphate,arylhalides (difluoro-dinitrobenzene), anhydride, alkyl halide, or acidhalide, p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenylester, pentafluorophenyl ester, carbonyl imidazole, carbonyl pyridinium,or carbonyl dimethylaminopyridinium, then R9,R14 can react with (but notrestricted to) an amine, a hydroxyl, hydrazine, hydrazide, or sulfhydrylgroup.

If functional group R9,R14 an activated carboxylic acid, haloacetylderivative, maleimide, aziridine derivative, acryloyl derivative,fluorobenzene derivatives, or disulfide derivative (such as a pyridyldisulfide or 5-thio-2-nitrobenzoic acid {TNB} derivatives) then R9,R14can react with (but not restricted to) a sulfhydryl.

If functional group R9,R14 is an aldehyde, ketone, epoxide, oxirane, oran amine in which carbonyldiimidazole or N,N′-disuccinimidyl carbonateis used, then R9,R14 can react with (but not restricted to) a hydroxyl.

If functional group R9,R14 is a hydrazine, hydrazide derivative, oramine (primary or secondary) then R9,R14 can react with (but notrestricted to) an aldehyde or ketone (to form a Schiff Base that may ormay not be reduced by reducing agents such as NaCNBH₃).

DETAILED DESCRIPTION

Counterintuitive to previous efforts to synthesize bifunctionalmolecules with stabile disulfides, the object of the current inventionis to synthesize labile disulfide molecules. In vivo, disulfides areprimarily reduced by the cysteine-based thiol glutathione(γ-glutamylcystylglycine), which is present in millimolar concentrationsin the cell. To increase the lability of the disulfide bond in abifunctional molecule and its construct, we have synthesized severaldisulfide bond-containing bifunctional molecules that are more rapidlyreduced than oxidized glutathione.

Disulfide Bond Containing Bifunctional Molecules

Bifunctional molecules, possessing either homo or heterobifunctionality(commonly referred to as crosslinkers), are used to connect twomolecules together. The disulfide linkage (RSSR′) may be used withinbifunctional molecules. The reversibility of disulfide bond formationmakes them useful tools for the transient attachment of two molecules.Physiologically, disulfides are reduced by glutathione.

A disulfide bond that is labile under physiological conditions means:the disulfide bond is cleaved more rapidly than oxidized glutathione orany disulfide constructed from thiols in which one of the constituentthiols is more acidic, lower pKa, than glutathione or is activated byintramolecular attack by a free thiol. Constituent in this case meansthe thiols that are bonded together in the disulfide bond. Cleavablemeans that a chemical bond between atoms is broken.

The present invention describes physiologically labile disulfide bondcontaining bifunctional molecules. The present invention is also meantto include constructs prepared from the bifunctional molecules,including polymers, peptides, proteins, nucleic acids, polymer nucleicacid complexes. Construct means any compound resulting from the chemicalreaction of at least one of the reactive centers of the bifunctionalmolecule resulting in new chemical bond other that that resulting fromhydrolysis of both reactive centers of the bifunctional molecule.Further chemical modification may occur after the formation of theconstruct. Crosslinking refers to the chemical attachment of two or moremolecules with a bifunctional reagent. A bifunctional reagent is amolecule with two reactive ends. The reactive ends can be identical asin a homobifunctional molecule, or different as in a heterobifunctionalmolecule.

Polymers

A polymer is a molecule built up by repetitive bonding together ofsmaller units called monomers. In this application the term polymerincludes both oligomers which have two to about 80 monomers and polymershaving more than 80 monomers. The polymer can be linear, branchednetwork, star, comb, or ladder types of polymer. The polymer can be ahomopolymer in which a single monomer is used or can be copolymer inwhich two or more monomers are used. Types of copolymers includealternating, random, block and graft.

To those skilled in the art of polymerization, there are severalcategories of polymerization processes that can be utilized in thedescribed process. The polymerization can be chain or step. Thisclassification description is more often used that the previousterminology of addition and condensation polymer. “Most step-reactionpolymerizations are condensation processes and most chain-reactionpolymerizations are addition processes” (M. P. Stevens PolymerChemistry: An Introduction New York Oxford University Press 1990).Template polymerization can be used to form polymers from daughterpolymers.

Step Polymerization: In step polymerization, the polymerization occursin a stepwise fashion. Polymer growth occurs by reaction betweenmonomers, oligomers and polymers. No initiator is needed since there isthe same reaction throughout and there is no termination step so thatthe end groups are still reactive. The polymerization rate decreases asthe functional groups are consumed.

Typically, step polymerization is done either of two different ways. Oneway, the monomer has both reactive functional groups (A and B) in thesame molecule so that A-B yields -[A-B]- Or the other approach is tohave two bifunctional monomers. A—A+B—B yields -[A—A-B-B]- Generally,these reactions can involve acylation or alkylation. Acylation isdefined as the introduction of an acyl group (—COR) onto a molecule.Alkylation is defined as the introduction of an alkyl group onto amolecule. If functional group A is an amine then B can be (but notrestricted to) an isothiocyanate, isocyanate, acyl azide,N-hydroxysuccinimide, sulfonyl chloride, aldehyde (includingformaldehyde and glutaraldehyde), ketone, epoxide, carbonate,imidoester, carboxylate activated with a carbodiimide, alkylphosphate,arylhalides (difluoro-dinitrobenzene), anhydride, or acid halide,p-nitrophenyl ester, o-nitrophenyl ester, pentachlorophenyl ester,pentafluorophenyl ester, carbonyl imidazole, carbonyl pyridinium, orcarbonyl dimethylaminopyridinium. In other terms when function A is anamine then function B can be acylating or alkylating agent or aminationagent.

If functional group A is a sulfhydryl then function B can be (but notrestricted to) an iodoacetyl derivative, maleimide, aziridinederivative, acryloyl derivative, fluorobenzene derivatives, or disulfidederivative (such as a pyridyl disulfide or 5-thio-2-nitrobenzoicacid{TNB} derivatives).

If functional group A is carboxylate then function B can be (but notrestricted to) a diazoacetate or an amine in which a carbodiimide isused. Other additives may be utilized such as carbonyldiimidazole,dimethylamino pyridine (DMAP), N-hydroxysuccinimide or alcohol usingcarbodiimide and DMAP.

If functional group A is an hydroxyl then function B can be (but notrestricted to) an epoxide, oxirane, or an amine in whichcarbonyldiimidazole or N,N′-disuccinimidyl carbonate, orN-hydroxysuccinimidyl chloroformate or other chloroformates are used. Iffunctional group A is an aldehyde or ketone then function B can be (butnot restricted to) an hydrazine, hydrazide derivative, amine (to form aSchiff Base that may or may not be reduced by reducing agents such asNaCNBH3) or hydroxyl compound to form a ketal or acetal.

Yet another approach is to have one bifunctional monomer so that A—Aplus another agent yields -[A-A]-.

If function A is a sulfhydryl group then it can be converted todisulfide bonds by oxidizing agents such as iodine (I2) or NaIO4 (sodiumperiodate), or oxygen (O2). Function A can also be an amine that isconverted to a sulfhydryl group by reaction with 2-Iminothiolate(Traut's reagent) which then undergoes oxidation and disulfideformation. Disulfide derivatives (such as a pyridyl disulfide or5-thio-2-nitrobenzoic acid {TNB} derivatives) can also be used tocatalyze disulfide bond formation. Functional group A or B in any of theabove examples could also be a photoreactive group such as aryl azide(including halogenated aryl azide), diazo , benzophenone, alkyne ordiazirine derivative.

Reactions of the amine, hydroxyl, sulfhydryl, carboxylate groups yieldchemical bonds that are described as amide, amidine, disulfide, ethers,esters, enamine, imine, urea, isothiourea, isourea, sulfonamide,carbamate, alkylamine bond (secondary amine), carbon-nitrogen singlebonds in which the carbon contains a hydroxyl group, thioether, diol,hydrazone, diazo, or sulfone.

Chain Polymerization: In chain-reaction polymerization growth of thepolymer occurs by successive addition of monomer units to limited numberof growing chains. The initiation and propagation mechanisms aredifferent and there is usually a chain-terminating step. Thepolymerization rate remains constant until the monomer is depleted.Monomers containing (but not limited to) vinyl, acrylate, methacrylate,acrylamide, methacrylamide groups can undergo chain reaction which canbe radical, anionic, or cationic. Chain polymerization can also beaccomplished by cycle or ring opening polymerization. Several differenttypes of free radical initiators could be used that include peroxides,hydroxy peroxides, and azo compounds such as2,2′-Azobis(-amidinopropane) dihydrochloride (AAP).

Types of Monomers

A wide variety of monomers can be used in the polymerization processes.These include positive charged organic monomers such as amine salts,imidine, guanidine, imine, hydroxylamine, hydrozyine, heterocycle(salts) like imidazole, pyridine, morpholine, pyrimidine, or pyrene. Theamines could be pH-sensitive in that the pKa of the amine is within thephysiologic range of 4 to 8. Specific amines include spermine,spermidine, N,N′-bis(2-aminoethyl)-1,3-propanediamine (AEPD), and3,3′-Diamino-N,N-dimethyldipropylammonium bromide.

Monomers can also be hydrophobic, hydrophilic or amphipathic.Amphipathic compounds have both hydrophilic (water-soluble) andhydrophobic (water-insoluble) parts. Hydrophilic groups indicate inqualitative terms that the chemical moiety is water-preferring.Typically, such chemical groups are water soluble, and are hydrogen bonddonors or acceptors with water. Examples of hydrophilic groups includecompounds with the following chemical moieties; carbohydrates,polyoxyethylene, peptides, oligonucleotides and groups containingamines, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls.Hydrophobic groups indicate in qualitative terms that the chemicalmoiety is water-avoiding. Typically, such chemical groups are not watersoluble, and tend not to hydrogen bonds. Hydrocarbons are hydrophobicgroups.

Monomers can also be intercalating agents such as acridine, thiazoleorgange, or ethidium bromide. Monomers can also contain chemicalmoieties that can be modified before or after the polymerizationincluding (but not limited to) amines (primary, secondary, andtertiary), amides, carboxylic acid, ester, hydroxyl, hydrazine, alkylhalide, aldehyde, and ketone.

Other Components of the Monomers and Polymers

The polymers have other groups that increase their utility. These groupscan be incorporated into monomers prior to polymer formation or attachedto the polymer after its formation. These groups include: targetinggroups, signals, reporter or marker molecules, spacers, stericstabilizers, chelators, polycations, polyanions, and polymers.

Targeting groups are used for targeting the polymer-nucleic acidcomplexes to specific cells or tissues. Examples of targeting agentsinclude agents that target to the asialoglycoprotein receptor by usingasiologlycoproteins or galactose residues. Proteins such as insulin,EGF, or transferrin can be used for targeting. Protein refers to amolecule made up of 2 or more amino acid residues connected one toanother by peptide bonds between the alpha-amino group and carboxylgroup of contiguous amino acid residues as in a polypeptide. The aminoacids may be naturally occurring or synthetic. Peptides that include theRGD sequence can be used to target many cells. Peptide refers to alinear series of amino acid residues connected to one another by peptidebonds between the alpha-amino group and carboxyl group of contiguousamino acid residues. Polypeptide includes proteins and peptides,modified proteins and peptides, and non-natural proteins and peptides.

Chemical groups that react with sulfhydryl or disulfide groups on cellscan also be used to target many types of cells. Folate and othervitamins can also be used for targeting. Other targeting groups includemolecules that interact with membranes such as fatty acids, cholesterol,dansyl compounds, and amphotericin derivatives.

Other targeting groups can be used to increase the delivery of the drugor nucleic acid to certain parts of the cell. For example, agents can beused to disrupt endosomes and a nuclear localizing signal (NLS) can beused to target the nucleus. A variety of ligands have been used totarget drugs and genes to cells and to specific cellular receptors. Theligand may seek a target within the cell membrane, on the cell membraneor near a cell. Binding of ligands to receptors typically initiatesendocytosis. Ligands could also be used for DNA delivery that bind toreceptors that are not endocytosed. For example peptides containing RGDpeptide sequence that bind integrin receptor could be used. In additionviral proteins could be used to bind the complex to cells. Lipids andsteroids could be used to directly insert a complex into cellularmembranes. The polymers can also contain cleavable groups withinthemselves. When attached to the targeting group, cleavage leads toreduce interaction between the complex and the receptor for thetargeting group. Cleavable groups include but are not restricted todisulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds,acetals, ketals, enol ethers, enol esters, enamines and imines, acylhydrazones, and Schiff bases.

In a preferred embodiment, a chemical reaction can be used to attach asignal to a nucleic acid complex. The signal is defined in thisspecification as a molecule that modifies the nucleic acid complex, orbiologically active molecule, and can direct it to a cell location (suchas tissue cells) or location in a cell (such as the nucleus, orcytoplasm) either in culture or in a whole organism. By modifying thecellular or tissue location of the foreign gene, the expression of theforeign gene can be enhanced.

The signal can be a protein, peptide, lipid, steroid, sugar,carbohydrate, nucleic acid or synthetic compound. The signals enhancecellular binding to receptors, cytoplasmic transport to the nucleus andnuclear entry or release from endosomes or other intracellular vesicles.

A certain subset of signals, termed transduction signals in thisapplication, transport themselves and attached molecules acrossmembranes (Schwarze and Dowdy Trends

Pharm. Sci. 2000, 21, 45). Examples of these transduction signals arederived from viral coat proteins such as Tat from HJV and VP22 fromherpes simplex virus, and a transcriptional factor from Drosophila,ANTP. The peptides Tat (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg, SEQID NO 1), VP22(Asp-Ala-Ala-Thr-Ala-Thr-Arg-Gly-Arg-Ser-Ala-Ala-Ser-Arg-Pro-Thr-Glu-Arg-Pro-Arg-Ala-Pro-Ala-Arg-Ser-Ala-Ser-Arg-Pro-Arg-Arg-Pro-Val-Glu,SEQ ID NO 2), and ANTP(Arg-Gln-Iso-Lys-Iso-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys, SEQ IDNO 3) share no sequence motif other than number of cationic (lysine andarginine) residues. In addition, reports of synthetic peptidespossessing no homology other than a propensity of cationic charge (netoverall cationic charge) have also been shown to posses transductionactivity (Service, R.F. Science 2000, 288, 28.)

Nuclear localizing signals enhance the targeting of the gene intoproximity of the nucleus and/or its entry into the nucleus. Such nucleartransport signals can be a protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear localizing signalsinteract with a variety of nuclear transport factors such as the NLSreceptor (karyopherin alpha) which then interacts with karyopherin beta.The nuclear transport proteins themselves could also function as NLS'ssince they are targeted to the nuclear pore and nucleus.

Signals that enhance release from intracellular compartments (releasingsignals) can cause DNA release from intracellular compartments such asendosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmicreticulum, golgi apparatus, trans golgi network (TGN), and sarcoplasmicreticulum. Release includes movement out of an intracellular compartmentinto cytoplasm or into an organelle such as the nucleus. Releasingsignals include chemicals such as chioroquine, bafilomycin or BrefeldinAl and the ER-retaining signal (KDEL sequence. SEQ ID NO 4), viralcomponents such as influenza virus hemagglutinin subunit HA-2 peptidesand other types of amphipathic peptides. Cellular receptor signals areany signal that enhances the association of the gene or particle with acell. This can be accomplished by either increasing the binding of thegene to the cell surface and/or its association with an intracellularcompartment, for example: ligands that enhance endocytosis by enhancingbinding the cell surface. This includes agents that target to theasialoglycoprotein receptor by using asiologlycoproteins or galactoseresidues. Other proteins such as insulin, EGF, or transferrin can beused for targeting. Peptides that include the RGD sequence can be usedto target many cells. Chemical groups that react with sulfhydryl ordisulfide groups on cells can also be used to target many types ofcells. Folate and other vitamins can also be used for targeting. Othertargeting groups include molecules that interact with membranes such aslipids fatty acids, cholesterol, dansyl compounds, and amphotericinderivatives. In addition viral proteins could be used to bind cells.

Reporter or marker molecules are compounds that can be easily detected.Typically they are fluorescent compounds such as fluorescein, rhodamine,Texas red, cy 5, cy 3 or dansyl compounds. They can be molecules thatcan be detected by UV or visible spectroscopy or by antibodyinteractions or by electron spin resonance. Biotin is another reportermolecule that can be detected by labeled avidin. Biotin could also beused to attach targeting groups.

A spacer is any linker known to those skilled in the art to enable oneto join one moiety to another moiety. The moieties can be hydrophilic orhydrophobic. Preferred spacer groups include, but are not limited toC1–C12 alkyl, C1–C12 alkenyl, C1-C12 alkynyl, C6–C18 aralkyl, C6–C 18aralkenyl, C6–C 18 aralkynyl, ester, ether, ketone, alcohol, polyol,amide, amine, polyglycol, polyamine, thiol, thio ether, thioester,phosphorous containing, and heterocyclic.

A Steric stabilizer is a long chain hydrophilic group that preventsaggregation of final polymer by sterically hindering particle toparticle electrostatic interactions. Examples include: alkyl groups, PEGchains, polysaccharides, hydrogen molecules, alkyl amines. Electrostaticinteractions are the non-covalent association of two or more substancesdue to attractive forces between positive and negative charges.

A polycation is a polymer containing a net positive charge, for examplepoly-L-lysine hydrobromide. The polycation can contain monomer unitsthat are charge positive, charge neutral, or charge negative, however,the net charge of the polymer must be positive. A polycation also canmean a non-polymeric molecule that contains two or more positivecharges. A polyanion is a polymer containing a net negative charge, forexample polyglutamic acid. The polyanion can contain monomer units thatare charge negative, charge neutral, or charge positive, however, thenet charge on the polymer must be negative. A polyanion can also mean anon-polymeric molecule that contains two or more negative charges. Theterm polyion includes polycation, polyanion, zwitterionic polymers, andneutral polymers. The term zwitterionic refers to the product (salt) ofthe reaction between an acidic group and a basic group that are part ofthe same molecule. Salts are ionic compounds that dissociate intocations and anions when dissolved in solution. Salts increase the ionicstrength of a solution, and consequently decrease interactions betweennucleic acids with other cations.

A chelator is a polydentate ligand, a molecule that can occupy more thanone site in the coordination sphere of an ion, particularly a metal ion,primary amine, or single proton. Examples of chelators include crownethers, cryptates, and non-cyclic polydentate molecules. A crown etheris a cyclic polyether containing (—X—(CR1-2)n)m units, where n=1–3 andm=3–8. The X and CR1-2 moieties can be substituted, or at a differentoxidation states. X can be oxygen, nitrogen, or sulfur, carbon,phosphorous or any combination thereof. R can be H, C, O, S, N, P. Asubset of crown ethers described as a cryptate contain a second(—X—(CR1-2)n)z strand where z=3–8. The beginning X atom of the strand isan X atom in the (—X—(CR1-2)n)m unit, and the terminal CH2 of the newstrand is bonded to a second X atom in the (—X—(CR1-2)n)m unit.Non-cyclic polydentate molecules containing (—X—(CR1-2)n)m unit(s),where n=1–4 and m=1–8. The X and CR1-2 moieties can be substituted, orat a different oxidation states. X can be oxygen, nitrogen, or sulfur,carbon, phosphorous or any combination thereof. A polychelator is apolymer associated with a plurality of chelators by an ionic or covalentbond and can include a spacer. The polymer can be cationic, anionic,zwitterionic, neutral, or contain any combination of cationic, anionic,zwitterionic, or neutral groups with a net charge being cationic,anionic or neutral, and may contain steric stabilizers, peptides,proteins, signals, or amphipathic compound for the formation ofmicellar, reverse micellar, or unilamellar structures. Preferably theamphipathic compound can have a hydrophilic segment that is cationic,anionic, or zwitterionic, and can contain polymerizable groups, and ahydrophobic segment that can contain a polymerizable group.

The present invention provides for the transfer of polynucleotides, andbiologically active compounds into parenchymal cells within tissues insitu and in vivo, utilizing disulfide bonds that can be cleaved underphysialogicval condidtions, and delivered intravasculary (U.S. patentapplication Ser. No. 08/571,536), intrarterially, intravenous, orally,intraduodenaly, via the jejunum (or ileum or colon), rectally,transdermally, subcutaneously, intramuscularly, intraperitoneally,intraparenterally, via direct injections into tissues such as the liver,lung, heart, muscle, spleen, pancreas, brain (includingintraventricular), spinal cord, ganglion, lymph nodes, lymphatic system,adipose tissues, thryoid tissue, adrenal glands, kidneys, prostate,blood cells, bone marrow cells, cancer cells, tumors, eye retina, viathe bile duct, or via mucosal membranes such as in the mouth, nose,throat, vagina or rectum or into ducts of the salivary or other exocrineglands.

“Delivered” means that the polynucleotide becomes associated with thecell. The polynucleotide can be on the membrane of the cell or insidethe cytoplasm, nucleus, or other organelle of the cell. The process ofdelivering a polynucleotide to a cell has been commonly termed“transfection” or the process of “transfecting” and also it has beentermed “transformation”. The polynucleotide could be used to produce achange in a cell that can be therapeutic. The delivery ofpolynucleotides or genetic material for therapeutic and researchpurposes is commonly called “gene therapy”. The polynucleotides orgenetic material being delivered are generally mixed with transfectionreagents prior to delivery.

A biologically active compound is a compound having the potential toreact with biological components. More particularly, biologically activecompounds utilized in this specification are designed to change thenatural processes associated with a living cell. For purposes of thisspecification, a cellular natural process is a process that isassociated with a cell before delivery of a biologically activecompound. In this specification, the cellular production of, orinhibition of a material, such as a protein, caused by a human assistinga molecule to an in vivo cell is an example of a delivered biologicallyactive compound. Pharmaceuticals, proteins, peptides, polypeptides,hormones, cytokines, antigens, viruses, oligonucleotides, and nucleicacids are examples of biologically active compounds. Bioactive compoundsmay be used interchangeably with biologically active compound forpurposes of this application.

The term “nucleic acid” is a term of art that refers to a polymercontaining at least two nucleotides. “Nucleotides” contain a sugardeoxyribose (DNA) or ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through the phosphate groups. “Bases”include purines and pyrimidines, which further include natural compoundsadenine, thymine, guanine, cytosine, uracil, inosine, and syntheticderivatives of purines and pyrimidines, or natural analogs. Nucleotidesare the monomeric units of nucleic acid polymers. A “polynucleotide” isdistinguished here from an “oligonucleotide” by containing more than 80monomeric units; oligonucleotides contain from 2 to 80 nucleotides. Theterm nuclei acid includes deoxyribonucleic acid (DNA) and ribonucleicacid (RNA). DNA may be in the form of anti-sense, plasmid DNA, parts ofa plasmid DNA, vectors (P1, PAC, BAC, YAC, artificial chromosomes),expression cassettes, chimeric sequences, chromosomal DNA, orderivatives of these groups. RNA may be in the form of oligonucleotideRNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomalRNA), mRNA (messenger RNA), anti-sense RNA, ribozymes, chimericsequences, or derivatives of these groups. “Anti-sense” is apolynucleotide that interferes with the function of DNA and/or RNA. Thismay result in suppression of expression. Natural nucleic acids have aphosphate backbone, artificial nucleic acids may contain other types ofbackbones and bases. These include PNAs (peptide nucleic acids),phosphothionates, and other variants of the phosphate backbone of nativenucleic acids.

In addition, DNA and RNA may be single, double, triple, or quadruplestranded. “Expression cassette” refers to a natural or recombinantlyproduced polynucleotide molecule which is capable of expressingprotein(s). A DNA expression cassette typically includes a promoter(allowing transcription initiation), and a sequence encoding one or moreproteins. Optionally, the expression cassette may include trancriptionalenhancers, non-coding sequences, splicing signals, transcriptiontermination signals, and polyadenylation signals. An RNA expressioncassette typically includes a translation initiation codon (allowingtranslation initiation), and a sequence encoding one or more proteins.Optionally, the expression cassette may include translation terminationsignals, a polyadenosine sequence, internal ribosome entry sites (IRES),and non-coding sequences.

The term “naked polynucleotides” indicates that the polynucleotides arenot associated with a transfection reagent or other delivery vehiclethat is required for the polynucleotide to be delivered to the cardiacmuscle cell. A “transfection reagent” or “delivery vehicle” is acompound or compounds used in the prior art that bind(s) to orcomplex(es) with oligonucleotides or polynucleotides, and mediates theirentry into cells. The transfection reagent also mediates the binding andinternalization of polynucleotides into cells. Examples of transfectionreagents include cationic liposomes and lipids, polyamines, calciumphosphate precipitates, histone proteins, polyethylenimine, andpolylysine complexes (polyethylenimine and polylysine are both toxic).Typically, the transfection reagent has a net positive charge that bindsto the polynucleotide's negative charge. The transfection reagentmediates binding of polynucleotides to cell via its positive charge(that binds to the cell membrane's negative charge) or via ligands thatbind to receptors in the cell. For example, cationic liposomes orpolylysine complexes have net positive charges that enable them to bindto DNA or RNA. Other delivery vehicles are also used, in the prior art,to transfer genes into cells. These include complexing thepolynucleotides on particles that are then accelerated into the cell.This is termed “biolistic” or “gun” techniques.

Ionic (electrostatic) interactions are the non-covalent association oftwo or more substances due to attractive forces between positive andnegative charges, or partial positive and partial negative charges.

Condensed Nucleic Acids: A method of condensing a polymer is defined asdecreasing its linear length, also called compacting. Condensing apolymer also means decreasing the volume that the polymer occupies. Anexample of condensing nucleic acid is the condensation of DNA thatoccurs in cells. The DNA from a human cell is approximately one meter inlength but is condensed to fit in a cell nucleus that has a diameter ofapproximately 10 microns. The cells condense (or compacts) DNA by aseries of packaging mechanisms involving the histones and otherchromosomal proteins to form nucleosomes and chromatin. The DNA withinthese structures is rendered partially resistant to nuclease DNase)action. The process of condensing polymers can be used for deliveringthem into cells of an organism. A delivered polymer can stay within thecytoplasm or nucleus apart from the endogenous genetic material.Alternatively, the polymer could recombine (become a part of) theendogenous genetic material. For example, DNA can insert intochromosomal DNA by either homologous or non-homologous recombination.

Intravascular: An intravascular route of administration enables apolymer or polynucleotide to be delivered to cells more evenlydistributed and more efficiently expressed than direct injections.Intravascular herein means within a tubular structure called a vesselthat is connected to a tissue or organ within the body. Within thecavity of the tubular structure, a bodily fluid flows to or from thebody part. Examples of bodily fluid include blood, lymphatic fluid, orbile. Examples of vessels include arteries, arterioles, capillaries,venules, sinusoids, veins, lymphatics, and bile ducts. The intravascularroute includes delivery through the blood vessels such as an artery or avein. An administration route involving the mucosal membranes is meantto include nasal, bronchial, inhalation into the lungs, or via the eyes.

Buffers are made from a weak acid or weak base and their salts. Buffersolutions resist changes in pH when additional acid or base is added tothe solution. Biological, chemical, or biochemical reactions involve theformation or cleavage of ionic and/or covalent bonds. Biomolecule refersto peptides, polypeptides, proteins, enzymes, polynucleotides,oligonucleotides, viruses, antigens, carbohydrates (and conjugates),lipids, and saccharides. Enzymes are proteins evolved by the cells ofliving organisms for the specific function of catalyzing chemicalreactions. A chemical reaction is defined as the formation or cleavageof covalent or ionic bonds. As a result of the chemical reaction apolymer can be formed. A polymer is defined as a compound containingmore than two monomers. A monomer is a compound that can be attached toitself or another monomer and thus a form a polymer.

Transdermal refers to application to mammal skin in which drug deliveryoccurs by crossing the dermal layer.

Hydrocarbon means containing carbon and hydrogen atoms; andhalohydrocarbon means containing carbon, halogen (F, Cl, Br, I), andhydrogen atoms.

Alkyl means containing sp³ hybridized carbon atoms; alkenyl meanscontaining two or more sp² hybridized carbon atoms; aklkynyl meanscontaining two or more sp hybridized carbon atoms; aralkyl meanscontaining one or more aromatic ring(s) in addition containing sp³hybridized carbon atoms; aralkenyl means containing one or more aromaticring(s) in addition to containing two or more sp² hybridized carbonatoms; aralkynyl means containing one or more aromatic ring(s) inaddition to containing two or more sp hybridized carbon atoms; steroidincludes natural and unnatural steroids and steroid derivatives.

A steroid derivative means a sterol, a sterol in which the hydroxylmoity has been modified (for example, acylated), or a steroid hormone,or an analog thereof.

Carbohydrates include natural and unnatural sugars (for exampleglucose), and sugar derivatives (a sugar derivative means a system inwhich one or more of the hydroxyl groups on the sugar moiety has beenmodified (for example acylated), or a system in which one or more of thehydroxyl groups is not present).

Polyoxyethylene means a polymer having two to six (n=2–3000) ethyleneoxide units (—(CH₂CH₂O)_(n)—) or a derivative thereof.

R is meant to be any compatible group, for example hydrogen, alkyl,alkenyl, alkynyl, aralkyl, aralkenyl, or aralkynyl, and can includeheteroatoms (N, O, S), and carbonyl groups.

A compound is a material made up of two or more elements.

Electron withdrawing group is any chemical group or atom composed ofelectronegative atom(s), that is atoms that tend to attract electrons.Resonance stabilization is the ability to distribute charge on multipleatoms through pi bonds. The inductive effective, in a molecule, is ashift of electron density due to the polarization of a bond by a nearbyelectronegative or electropositive atom.

Steric hindrance, or sterics, is the prevention or retardation of achemical reaction because of neighboring groups on the same molecule.

An activated carboxylate is a carboxylic acid derivative that reactswith nucleophiles to form a new covalent bond. Nucleophiles includenitrogen, oxygen and sulfur-containing compounds to produce ureas,amides, carbonates, esters, and thioesters. The carboxylic acid may beactivated by various agents including carbodiimides, carbonates,phosphoniums, uroniums to produce activated carboxylates acyl ureas,acylphosphonates, and carbonates. Activation of carboxylic acid may beused in conjunction with hydroxy and amine-containing compounds toproduce activated carboxylates N-hydroxysuccinimide esters,hydroxybenzotriazole esters,N-hydroxy-5-norbomene-endo-2,3-dicarboximide 2,3-dicarboximide esters,p-nitrophenyl esters, pentafluorophenyl esters,4-dimethylaminopyridinium amides, and acyl imidazoles.

A nucleophile is a species possessing one or more electron-rich sites,such as an unshared pair of electrons, the negative end of a polar bond,or pi electrons.

EXAMPLES cl Example 1 Synthesis of Cysteine-Terminal Tat Peptide(Tat-Cys).

Peptide syntheses were performed using standard solid phase peptidetechniques using FMOC chemistry. A cysteine was added to the aminoterminus of Tat to allow for conjugation through the thiol group to makethe peptide Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys (Tat-Cys.SEQ ID NO 5).

Example 2 Synthesis of noncleavably linked (irreversible covalent)Tat-Cys and fluorescein through a thioether bond.

To a solution of succinimidyl-4-(N-maleimidomethyl)cyclohexane-carboxylate (SMCC from Pierce) 1.0 mg in 0.1 mLdimethylformamide was added 1.2 mg (1 eq) of4′-(aminomethyl)fluorescein. After two hours, this solution was added toa 1 mL aqueous solution of 8.4 mg Tat-Cys (1 eq). The solution wasbuffered to pH 8 by the addition of potassium carbonate. This solutionwas used for transport studies without further purification.

Example 3 Synthesis of Unactivated (Non-labile) Disulfide LinkedLissamine Dimer (Dilissamine Cystamine)

To a solution of cystamine dihydrochloride (10 mg) in water (1 mL) wasadded diisopropylethylamine (15 μL, 2 eq). To this was added lissaminechloride (Rhodamine B sulfonyl chloride, Molecular Probes) 77 mg (3 eq)in 5 mL of methanol. The solution was stirred for 1 hour and thenchromatographed by reverse-phase HPLC using an Aquasil C-18 column usinga gradient from 100% 0.1% trifluoroacetic acid in water to 100% 0.1%triflouroacetic acid in acetonitrile. The fraction containing theproduct was determined by mass spectroscopy. The molecular weight ofcompound is 1234, which was detected in positive ion mode. Theconcentration of the product-containing fraction was determined by theabsorbance of the solution at 588 nm (ε=88,000 M⁻¹ cm⁻¹).

Example 4 Attachment of Lissamine to Tat-Cys by an Unactivated Disulfide

To a solution of Tat-Cys (100 μg) in 100 μL water was added dilissaminecystamine (41 μg, 1 eq). The pH of the solution was adjusted to 7–8 bythe addition of potassium carbonate.

Example 5 Synthesis of Activated (Labile) Disulfide-containing LissamineAdduct (Lissamine 4-aminophenyl Disulfide)

To a solution of 10 mg of lissamine chloride (Rhodamine B sulfonylchloride, Molecular Probes) in 0.2 mL dimethylformamide was added overfive minutes ten 10 μL aliquots of 4-aminophenyl disulfide (2 mg, 0.5eq) and diisopropylethylamine (3 μL, 1 eq). Two hours after finaladdition of disulfide the solution was diluted into 2 mL of acetonitrileand chromatographed by reverse-phase HPLC using an Aquasil C-18 columnapplying a gradient from 20% acetonitrile and 80% water containing 0.1%trifluoroacetic acid to 100% 0.1% triflouroacetic acid in acetonitrile.We were unable to isolate the lissamine dimer, but were able to isolatethe product of monoaddition. The fraction containing the monoadditionproduct was determined by mass spectroscopy. The molecular weight ofcompound is 789, which was detected in positive ion mode. Theconcentration of the product-containing fraction was determined by theabsorbance of the solution at 588 nm (ε=88,000 M⁻¹ cm⁻¹).

Example 6 Attachment of Lissamine to Tat-Cys by an Activated Disulfide

To a solution of Tat-Cys (100 μg) in 100 μL water was added Lissamine4-aminophenyl disulfide (26 μg, 1 eq). The pH of the solution wasadjusted to 7–8 by the addition of potassium carbonate.

Example 7 Synthesis of Activated (Labile) Disulfide Fluorescein Dimer(Difluorescein 4-aminophenyl Disulfide)

To a solution of 20 mg of fluorescein isothiocyanate in 0.5 mLdimethylformamide was added 4-aminophenyl disulfide (4 mg, 0.33 eq) in100 μL dimethylformamide and diisopropylethylamine (3 μL, 0.33 eq).After two hours, the solution was diluted into 2 mL of water that wasbrought to pH 8 with potassium carbonate. This aqueous solution wasfiltered and chromatographed by reverse-phase HPLC using an Aquasil C-18column applying a gradient from 100% water containing 0.1%trifluoroacetic acid to 100% 0.1% triflouroacetic acid in acetonitrile.The fraction containing the product was determined by mass spectroscopy.The molecular weight of compound is 1025, which was detected in negativeion mode. The concentration of the product-containing fraction wasdetermined by the absorbance of the solution at 494 nm (ε=75,000 M⁻¹cm⁻¹).

Example 8 Measurement of the Reduction of Unactivated (Non-labile)Disulfide (Dilissamine Cystamine).

To a solution containing 0.44 μM dilissamine cystamine and 100 mM sodiumphosphate pH 7.5 was added glutathione to a concentration of 250 μM. Thesolution was irradiated with 555 nm light and the fluorescence of thesolution was measured at 585 nm. The amount of time required to reachhalf maximum fluorescence was 2000–2400 sec.

Example 9 Measurement of the Reduction of Activated (Labile) Disulfide(Difluorescein 4-aminophenyl Disulfide).

To a solution containing 0.44 μM fluorescein 4-aminophenyl disulfide and100 mM sodium phosphate pH 7.5 was added glutathione to a concentrationof 250 μM. The solution was irradiated with 495 nm light and thefluorescence of the solution was measured at 520 nm. The amount of timerequired to reach half maximum fluorescence was 30–50 sec.

Example 10 Analysis of Delivery to Cells by TAT Peptide.

Grow HeLa cells on glass coverslips by incubating at 4° C. in Delbecco'sModified Eagle's Media (DMEM) supplemented with 50 μg TATpeptide-fluorophore chimera (pulse). At this temperature, endocytosis isbelieved to be completely inhibited. Incubate the cells for two hours at4° C. and then wash with DMEM to remove external TAT-fluorophore. Removethe media and then either process cells for fluorescence microscopy orincubate three more hours at 4° C. with DMEM with media changes everyhour (chase). The cells that are chased are then processed forfluorescence microscopy. Cells processed for fluorescence microscopy arewashed 3× in phosphate-buffered saline (PBS), fixed in PBS+4%formaldehyde for 20 min, washed 3× in PBS, and coverslips are mounted onslides.

The presence of fluorophore was detected by confocal microscopy (ZeissLSM 510). In the case of irreversible covalent thioether linkage betweenTAT and fluorophore, fluorescence was detected inside of the cell afterthe initial two hour incubation. Subsequent incubation of the cells withfluorophore-free media (chase) resulted in cells with no internalizedfluorophore. Similarly, TAT-fluorophore adducts linked through anunactivated disulfide cystamine bond also had initial internalizationthat disappeared upon incubation with chase solutions. For the activateddisulfide 4-aminophenyl disulfide, fluorescence was detected inside ofthe cell after the initial two hour incubation. In contrast to the otherattachments between flourophore and TAT, a chase of the fluorophore withfluorophore-free media did not show a reduction in the amount ofinternalized fluorophore.

Example 11 Synthesis of 5,5′-Dithiobis(2-nitrobenzoate)propionitrile

5,5′-dithiobis(2-nitrobenzoic acid) (500 mg, 1.26 mmol, Aldrich ChemicalCompany) was taken up in 4.0 mL dioxane. Dicylohexylcarbodiimide (540mg, 2.6 mmol, Aldrich Chemical Company) and 3-hydroxypropionitrile (240μL, 188 mg, 2.60 mmol, Aldrich Chemical Company) were added. Thereaction mixture was stirred overnight at room temperature. Theprecipitate was removed by centrifugation, and the solvent concentratedunder reduced pressure. The residue was washed with saturated sodiumbicarbonate, water, and brine; and dried over magnesium sulfate. Solventremoval (aspirator) yielded 696 mg yellow/orange foam. The residue waspurified using normal phase HPLC (Alltech econosil, 250×22 nm), flowrate=9.0 mL/min, mobile phase=1% ethanol in chloroform, retentiontime=13 min. Removal of solvent (aspirator) afforded 233 mg (36.8%) of5,5′-dithiobis(2-nitrobenzoate)propionitrile as a yellow oil. TLC(silica: 5% methanol in chloroform; Rf=0.51). H¹NMR ∂ 8.05 (d, 4 H),7.75 (m, 4H), 4.55 (t, 4H), 2.85 (t, 4H).

Example 12 Synthesis of Dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl

5,5′-Dithiobis(2-nitrobenzoate)propionitrile (113 mg, 0.226 mmol) wastaken up in 500. μL anhydrous chloroform. Anhydrous methanol (20.0 μL,0.494 mmol, Aldrich Chemical Company) was added. The resulting solutionwas cooled to 0° C. on an ice bath, and HCl gas was bubbled through thesolution for a period of 10 minutes. The resulting solution was placedin a −20° C. freezer for a period of 48 hours. During this time a yellowoil formed. The oil was washed thoroughly with chloroform and driedunder vacuum to afford 137 mg (95.8%) of dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl as a yellow foam.

Example 13 Polymerization of N-(2-Aminoethyl)-1,3-propanediamine andDimethyl 5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl on a DNATemplate.

Procedure:

Template polymerization was carried out in 25 mM HEPES buffer, pH 8.0.N-(2-Aminoethyl)-1,3-propanediamine (48 μg, 0.3 mM, Aldrich ChemicalCompany) was added to a 0.5 mL solution of pCIluc DNA (25 mg, 0.075 mMin phosphate, 2.6 μg/μL pCIluc; prepared according to Danko, I.,Williams, P., Herweijer, H. et al. Hum. Mol. Genetics (1997) in press).Dimethyl 5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl (500 μg,0.78 mM) was added, and the solution was vortexed. The reaction wasincubated at room temperature for one hour. A fine yellow precipitatewas observed to form during the incubation period. The reaction wascentrifuged to remove the precipitate. A portion of the reaction (10 μL)was reduced with 10 mM dithiothreitol (10 μL) to break the disulfidebonds forming the polymer. Portions (0.5 μg) of the intact polymer andthe reduced polymer were analyzed on a 1% agarose gel.

Example 14 Formation of DNA/Poly-L-Lysine/Dimethyl5,5′-Dithiobis(2-nitrobenzoate) propionimidate -2 HCl Complexes

pDNA/Poly-L-lysine hydrobromide complexes were prepared by combiningplasmid DNA (25 μg) with Poly-L-lysine hydrobromide (95 μg, MW 35 kDa,Aldrich Chemical Company) in 0.5 mL 25 mM Hepes buffer pH 8.0, and thesolution was vortexed to mix. The resulting solution was divided into 3portions. One portion was incubated at room temperature for 2 hrs. Tothe second portion was added dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl (472 mg, 1.5 mmol),the solution was mixed, and incubated at room temperature for 2 hrs. Tothe third sample was added dimethyl 3,3′-dithiobispropionimidate (1.1mg, 1.5 mmol), the solution was mixed, and incubated at room temperaturefor 2 hrs. After 2 hrs. the samples were then centrifuged at 12000 rpmfor five minutes.

Ninety degree light scattering measurements were performed (ShimadzoRF-1501 Fluorescence Spectrophotometer). The wavelength setting was 700nm for both the incident beam and detection of scattering light. Theslits for both beams were fixed at 10 nm. The particle size of theresulting complex was determined by light scattering (BrookhavenZetaPlus Particle Sizer). After determining the initial intensity ofscattered light, 15 μL 5 M NaCl solution was added to the complexeswhile the intensity of scattered light was monitored.

The addition of salt to the non-caged particles led to an immediateincrease in the turbidity of the solution indicating aggregation. Thenon-caged sample also became visibly cloudy. The addition of salt to theparticles caged using dimethyl 3,3′-dithiobispropionimidate led to anincrease in turbidity of approximately 33%. The addition of salt to thedimethyl 5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl cagedcomplexes lead to no visible rise in turbidity. The particle size of thedimethyl 5,5′-dithiobis(2-nitrobenzoate) propionimidate-2 HCl cagedparticles was determined (Brookhaven Zeta Plus Particle Sizer) in 150 mMNaCl (physiological concentration). The mean particle diameter was foundto be 89.7 nm, 67% of the total number of particles were under 100 nm insize.

The example indicates that dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl caged DNA. Theparticles formed are stable in physiological salt, and are under 100 nmin size.

Example 15 Demonstration of Reducibility of Disulfide Bond in vitro.

pDNA (pCI Luc)/polyethyleneimine (25 kDa, Aldrich ChemicalCompany)/dimethyl 3,3′-dithiobispropionimidate andpDNA/polyethyleneimine/dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl complexes wereprepared in 25 mM HEPES buffer pH 8.0. All complexes were prepared atpDNA/polyethyleneimine ratios of 1/3. Dimethyl3,3′-dithiobispropionimidate and dimethyl5,5′-dithiobis(2-nitrobenzoate) propionimidate-2 HCl were added at thefollowing ratios: 0, 3, 6, 12, and 25. Complexes were incubated 0.5 hourat room temperature, and centrifuged 5 minutes at 12,000 rpm prior totransfection. Transfections were carried out in 35 mm wells. At the timeof transfection, HepG2 monolayers, at approximately 50% confluency, werewashed once with PBS (phosphate buffered saline), and subsequentlystored in serum-free media (Opti-MEM, Gibco BRL). The complexes werediluted in Opti-MEM and added by drops, 5.0 μg DNA/well, to the cells.After a 4 hour incubation period at 37° C., the media containing thecomplexes was aspirated from the cells, and replaced with completegrowth media, DMEM with 10% fetal bovine serum (Sigma). After anadditional incubation of 42 hours, the cells were harvested and thelysate was assayed for luciferase expression (Wolff, J. A., Malone, R.W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Feigner, P. L.Direct gene transfer into mouse muscle in vivo. Science,1465–1468,1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany)luminometer was used.

pDNA/polyethyleneimine/methyl 3,3′-dithiobispropionimidate andpDNA/polyethyleneimine/dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl particles weretransfected into Hep G2 cells. pDNA/polyethyleneimine complexes werealso transfected as a control. The cell lysates were then analyzed forthe expression of luciferin. The results show that while the dimethyl3,3′-dithiobispropionimidate complexes gave expression results belowbaseline (<200 RLU), the dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2HCl/pDNA/polyethyleneimine complexes gave levels of expression that wereas high as 120,000 RLU.

The physiologically labile disulfide bonds present in the dimethyl5,5′-dithiobis(2-nitrobenzoate)propionimidate-2 HCl complexes can bereduced by cultured cells, while the disulfide bonds present in thedimethyl 3,3′-dithiobispropionimidate complexes cannot.

Example 16 Synthesis of 5,5′-dithiobis[(3″-bromopropyl)-2-nitrobenzoate]

5,5′-dithiobis-(2-nitrobenzoic acid) (500 mg, 1.26 mmol, AldrichChemical Company) and 3-bromopropanol (368 mg, 2.65 mmol, AldrichChemical Company) were taken up in 7.0 mL THF. Dicyclohexylcarbodiimide(545 mg, 2.65 mmol, Aldrich Chemical Company) was added, and thereaction mixture was stirred overnight at ambient temperature. Theprecipitate was removed by filtration, and the solution was concentratedunder reduced pressure to afford 430 mg (54%) of5,5′-dithiobis[(3″-bromopropyl)-2-nitrobenzoate] as a yellow oil.

Example 17 Synthesis of5,5′-dithiobis[(3″-ammonio-{N,N-dimethyl-N-propionitrile}propylbromide)2-nitrobenzoate]

5,5′-dithiobis[(3″-bromopropyl)-2-nitrobenzoate] was taken up in 2.0 mLTHF, and 3-dimethylaminopropionitrile (193 mg, 1.96 mmol, AldrichChemical Company) was added. After 3 days at ambient temperature, thesalt was precipitated from solution with Et₂O, and purified by reversephase HPLC (C-18 Aquasil 200×20 mm) using a gradient from 20 to 80%methanol over 20 minutes (elution at 15 minutes). The solvent wasremoved under reduced pressure to afford 15.2 mg (3%)5,5′-dithiobis[(3″-ammonio-{N,N-dimethyl-N-propionitrile}propylbromide)2-nitrobenzoate]. H¹-NMR (CD₃OD) ∂ 8.4–8.6 (m, 6H), 5.0 (t, 4H), 4.35 (t, 4H), 4.1 (m, 4H), 2.85 (m, 4H), 3. Synthesis of Dimethyl5,5′-dithiobis[(3″-ammonio-(N,N-dimethyl-N-propioimidate)propylchloride) 2-nitrobenzoate]-hydrochloride

Example 18 Synthesis of5,5′-dithiobis[(3″-ammonio-(N,N-dimethyl-N-propioimidate)propylchloride) 2-nitrobenzoate]

5,5′-dithiobis[(3″-ammonio-{N,N-dimethyl-N-propionitrile}propylbromide)2-nitrobenzoate] (15.2 mg, 0.018 mmol) was taken up in 1 mL ofmethanol. The solution was saturated with HCl at 0° C. The resultingsolution was held at −20° C. for 1 week. Et₂O was added and theprecipitate collected by filtration to afford 8.3 mg (47%) of dimethyl5,5′-dithiobis[(3″-ammonio-(N,N-dimethyl-N-propioimidate)propylchloride) 2-nitrobenzoate]-hydrochloride.

Example 19 Synthesis of N,N′-Bis(t-BOC)-L-cystine

To a solution of L-cystine (1 gm, 4.2 mmol, Aldrich Chemical Company) inacetone (10 mL) and water (10 mL) was added2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (2.5 gm, 10 mmol,Aldrich Chemical Company) and triethylamine (1.4 mL, 10 mmol, AldrichChemical Company). The reaction was allowed to stir overnight at roomtemperature. The water and acetone was then by rotary evaporationresulting in a yellow solid. The diBOC compound was then isolated byflash chromatography on silica gel eluting with ethyl acetate 0.1%acetic acid.

Example 20 Synthesis of L-cystine-1,4-bis(3-aminopropyl)piperazineCopolymer

To a solution of N,N′-Bis(t-BOC)-L-cystine (85 mg, 0.15 mmol) in ethylacetate (20 mL) was added N,N′-dicyclohexylcarbodiimide (108 mg, 0.5mmol) and N-hydroxysuccinimide (60 mg, 0.5 mmol). After 2 hr, thesolution was filtered through a cotton plug and1,4-bis(3-aminopropyl)piperazine (54 μL, 0.25 mmol) was added. Thereaction was allowed to stir at room temperature for 16 h. The ethylacetate was then removed by rotary evaporation and the resulting solidwas dissolved in trifluoroacetic acid (9.5 mL), water (0.5 mL) andtriisopropylsilane (0.5 mL). After 2 h, the trifluoroacetic acid wasremoved by rotary evaporation and the aqueous solution was dialyzed in a15,000 MW cutoff tubing against water (2×2 l) for 24 h. The solution wasthen removed from dialysis tubing, filtered through 5 μM nylon syringefilter and then dried by lyophilization to yield 30 mg of polymer.

Example 21 Synthesis of Guanidino-L-cystine

To a solution of cystine (1 gm, 4.2 mmol) in ammonium hydroxide (10 mL)in a screw-capped vial was added O-methylisourea hydrogen sulfate (1.8gm, 10 mmol). The vial was sealed and heated to 60° C. for 16 h. Thesolution was then cooled and the ammonium hydroxide was removed byrotary evaporation. The solid was then dissolved in water (20 mL),filtered through a cotton plug. The product was then isolated by ionexchange chromatography using Bio-Rex 70 resin and eluting withhydrochloric acid (100 mM).

Example 22 Synthesis ofGuanidino-L-cystine-L-1,4-bis(3-aminopropyl)piperazine Copolymer

To a solution of guanidino-L-cystine (64 mg, 0.2 mmol) in water (10 mL)was slowly added N,N′-dicyclohexylcarbodiimide (82 mg, 0.4 mmol) andN-hyroxysuccinimide (46 mg, 0.4 mmol) in dioxane (5 mL). After 16 hr,the solution was filtered through a cotton plug and1,4-bis(3-aminopropyl)piperazine (40 μL, 0.2 mmol) was added. Thereaction was allowed to stir at room temperature for 16 h and then theaqueous solution was dialyzed in a 15,000 MW cutoff tubing against water(2×2 l) for 24 h. The solution was then removed from dialysis tubing,filtered through 5 μM nylon syringe filter and then dried bylyophilization to yield 5 mg of polymer.

Example 23 The Particle Size ofpDNA-L-cystine-1,4-bis(3-aminopropyl)piperazine Copolymer andDNA-guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine CopolymerComplexes.

To a solution of pDNA (10 μg/mL) in 0.5 mL 25 mM HEPES buffer pH 7.5 wasadded 10 μg/mL L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer orguanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer. The sizeof the complexes between DNA and the polymers were measured. For bothpolymers, the size of the particles were approximately 60 nm.

Example 24 Condensation of DNA withL-cystine-1,4-bis(3-aminopropyl)piperazine Copolymer and Decondensationof DNA upon Addition of Glutathione

Fluorescein labeled DNA was used for the determination of DNAcondensation in complexes withL-cystine-1,4-bis(3-aminopropyl)piperazine copolymer. pDNA was modifiedto a level of 1 fluorescein per 100 bases using Mirus' LabelIt™Fluorescein kit. The fluorescence was determined using a fluorescencespectrophotometer (Shimadzu RF-1501 spectrofluorometer) at an excitationwavelength of 495 nm and an emission wavelength of 530 nm. (Trubetskoy,V. S., Slattum, P. M., Hagstrom, J. E., Wolff, J. A., Budker, V. G.,“Quantitative Assessment of DNA Condensation,” Anal. Biochem (1999)incorporated by reference).

The intensity of the fluorescence of the fluorescein-labeled DNA (10μg/mL) in 0.5 mL of 25 mM HEPES buffer pH 7.5 was 300 units. Uponaddition of 10 μg/mL of L-cystine-1,4-bis(3-aminopropyl)piperazinecopolymer, the intensity decreased to 100 units. To this DNA-polycationsample was added 1 mM glutathione and the intensity of the fluorescencewas measured. An increase in intensity was measured to the levelobserved for the DNA sample alone. The half life of this increase influorescence was 8 minutes.

The experiment indicates that DNA complexes with physiologically-labiledisulfide-containing polymers are cleavable in the presence of thebiological reductant glutathione.

Example 25 Mouse Tail Vein Injection ofDNA-L-cystine-1,4-bis(3-aminopropyl)piperazine Copolymer andDNA-guanidino-L-cystinel,4-bis(3-aminopropyl)piperazine CopolymerComplexes

Plasmid delivery in the tail vein of ICR mice was performed asdescribed. To PCILuc DNA (50 μg) in 2.5 mL H₂O was added eitherL-cystine-1,4-bis(3-aminopropyl)piperazine copolymer,guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer, orpoly-L-lysine (34,000 MW, Sigma Chemical Company) (50 μg). The sampleswere then injected into the tail vein of mice using a 30 gauge, 0.5 inchneedle. One day after injection, the animal was sacrificed, and aluciferase assay was conducted.

Polycation ng/liver poly-L-lysine 6.2L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer 439guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer 487

The experiment indicates that DNA complexes with thephysiologically-labile disulfide-containing polymers are capable ofbeing broken, thereby allowing the luciferase gene to be expressed.

Example 26 Rat Intramuscle injection ofDNA-L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer andDNA-guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymercomplexes.

Plasmid delivery intro rat leg was performed as described (Wolff, J. A.,Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. andFelgner, P. L. Direct gene transfer into mouse muscle in vivo. Science,1465–1468,1990.). To pCILuc DNA (100 μg/mL, 2.5 mL) was addedL-cystine-1,4-bis(3-aminopropyl)piperazine copolymer orguanidino-L-cystine1,4-bis(3-aminopropyl)piperazine copolymer (100μg/mL) and then injected into the leg muscles of a rat. After 7 days,the animal was sacrificed and a luciferase assay was conducted.

amount luciferase (ng) DNA complex per leg no polycation 3.3L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer 4.5guanidino-L-cystine1,4-bis(3-aminopropyl)piperazine 6.5 copolymer

The experiment indicates that DNA complexes with thephysiologically-labile disulfide-containing polymers are capable ofbeing broken, thereby allowing the luciferase gene to be expressed.

Example 27 Injection of DNA-L-cystine-1,4-bis(3-aminopropyl)piperazineCopolymer Complex and pDNA (pCI Luc)/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer Complex and pDNA (pCILuc)/5,5′-dithiobis(2-nitrobenzoicacid)-1,4-bis(3-aminopropyl)piperazine—Folate Copolymer Complexes intothe Intestinal Lumen of Mice.

Intestinal cells were transfected by injecting pDNA solutions into themesenteric vasculature. A 3-cm section of the small intestines wasclamped, blocking both vascular inflow and outflow. A volume of 250 μlcontaining 50 μg pCILuc and 50 μg poly(ethylenimine) (Aldrich ChemicalCo. MW 25,000 MW), L-cystine-1,4-bis(3-aminopropyl)piperazine copolymer,pDNA (pCI Luc)/5,5′-dithiobis(2-nitrobenzoicacid)-1,4-bis(3-aminopropyl)piperazine copolymer, and pDNA (pCILuc)/5,5′-dithiobis(2-nitrobenzoicacid)-1,4-bis(3-aminopropyl)piperazine-folate copolymer complexes wereinjected into the intestinal lumen of mice. After 3 minutes, the clampswere removed. One day after DNA delivery, the mice were sacrificed, theinjected section of the intestines was excised, cut in 3 cm sections andassayed for luciferase expression. Different areas of the intestineswere targeted (duodenum, jejunum, ileum).

Amount luciferase (pg) Complex Duodenum jejunum ileumDNA-poly(ethylenimine) 0.5 3.0 1.7 DNA-L-cystine-1,4-bis 6.2 3.7 2.8(3-aminopropyl)piperazine copolymer pDNA (pCILuc)/5,5′-dithiobis(2-nitro- 42 20 226 benzoicacid)-1,4-bis(3-aminopropyl) piperazine copolymer pDNA (pCILuc)/5,5′-dithiobis(2-nitro- 36 1.9 51 benzoicacid)-1,4-bis(3-aminopropyl) piperazine-folate copolymer

The experiment indicates that DNA complexes with labiledisulfide-containing polymers are capable of being broken, therebyallowing the luciferase gene to be expressed.

Example 28 Synthesis of 5,5′-Dithiobis[succinimidyl(2-nitrobenzoate)]

5,5′-dithiobis(2-nitrobenzoic acid) (50.0 mg, 0.126 mmol, AldrichChemical Company) and N-hyroxysuccinimide (29.0 mg, 0.252 mmol, AldrichChemical Company) were taken up in 1.0 mL dichloromethane.Dicylohexylcarbodiimide (52.0 mg, 0.252 mmol) was added and the reactionmixture was stirred overnight at room temperature. After 16 hr, thereaction mixture was partitioned in EtOAc/H₂O. The organic layer waswashed 2×H₂O, 1×brine, dried (MgSO₄) and concentrated under reducedpressure. The residue was taken up in CH₂Cl₂, filtered, and purified byflash column chromatography on silica gel (130×30 mm, EtOAc:CH₂Cl₂ 1:9eluent) to afford 42 mg (56%)5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] as a white solid. H¹NMR(DMSO) ∂ 7.81–7.77 (d, 2H), 7.57–7.26 (m, 4H), 3.69 (s, 8 H).

Example 29 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer

1,4-Bis(3-aminopropyl)piperazine (10 μL, 0.050 mmol, Aldrich ChemicalCompany) was taken up in 1.0 mL methanol and HCl (2 mL, 1 M in Et₂O,Aldrich Chemical Company) was added. Et₂O was added and the resultingHCl salt was collected by filtration. The salt was taken up in 1 mL DMFand 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (30 mg, 0.050 mmol)was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (35 μL, 0.20 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000–14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 23 mg (82%) of5,5′-dithiobis(2-nitrobenzoic acid)-1,4-bis(3-aminopropyl)piperazinecopolymer.

Example 30 Particle Sizing of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer Complexes

To 50 μg pDNA in 3 mL Ringers (0.85% sodium chloride, 0.03% potassiumchloride, 0.03% calcium chloride) was added 170 μg5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)pi Copolymer.Particle sizing (Brookhaven Instruments Coporation, ZetaPlus ParticleSizer, 190, 532 nm) indicated an effective diameter of 92 nm for thecomplex. A 50 μg pDNA in 3 mL Ringers sample indicated no particleformation.

5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-arninopropyl)piperazineCopolymer condenses pDNA, forming small particles.

Example 31 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer Complexes

Four complexes were prepared as follows:

-   Complex I: pDNA (pCI Luc, 200 μg) in 1 mL H₂O and diluted with 9 mL    Ringers prior to injection.-   Complex II: pDNA (pCI Luc, 200 μg) was mixed with poly-L-lysine (378    μg, MW 3400, Sigma Chemical Company) in 1 mL H₂O and diluted with 9    mL Ringers prior to injection.-   Complex III: pDNA (pCI Luc, 200 μg) was mixed with    5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)piperazine    Copolymer (400 μg) in 1 mL H₂O and diluted with 9 mL Ringers prior    to injection.-   Complex IV: pDNA (pCI Luc, 200 μg) was mixed with Histone H1 (1.2    mg, Sigma Chemical Company) in 1 mL H₂O and diluted with 9 mL    Ringers prior to injection.

2.5 mL and 250 μL tail vein injections of the complex were performed(Zhang, G., Budker, V., Wolff, J, High Levels of Foreign Gene Expressionin Hepatocytes from Tail Vein Injections of Naked Plasmid DNA. HumanGene Therapy, July, 1999, incorporated by reference). Results reportedare for liver expression. Luciferase expression was determined aspreviously reported (Wolff, J. A., Malone, R. W., Williams, P., Chong,W., Acsadi, G., Jani, A. and Felgner, P.L. Direct gene transfer intomouse muscle in vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&GBerthold, Bad-Wildbad, Germany) luminometer was used.

Results from 2.5 mL Injections

-   Complex I: 1,976,000-   Complex II: 128,000-   Complex III: 5,025,000-   Complex IV: 1,960    Results from 250 μL Injections-   Complex I: 985-   Complex III: 1,140

Results indicate an increased level of luciferase expression inpDNA/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer complexes over pCI LucDNA itself, pCI Luc DNA/poly-L-lysine complexes, and pCI Luc DNA/HistoneH1 complexes. These results also indicate that the pDNA is beingreleased from the pDNA/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer complexes, and isaccessible for transcription.

250 μL injection results were similar for bothpDNA/5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer complexes and pCI LucDNA.

Example 32 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine-Tris(2-aminoethyl)amine Copolymer

1,4-Bis(3-aminopropyl)piperazine (2.4 μL, 0.012 mmol, Aldrich ChemicalCompany) and tris(2-aminoethyl)amine (0.51 μL, 0.0034 mmol, AldrichChemical Company) were taken up in 0.5 mL methanol and HCl (1 mL, 1 M inEt₂O, Aldrich Chemical Company) was added Et₂O was added and theresulting HCl salt was collected by filtration.5,5′-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg, 0.016 mmol) wasadded and the mixture was taken up in 0.4 mL DMSO and 0.4 mL THF . Theresulting solution was stirred at room temperature anddiisopropylethylamine (5.9 μL, 0.042 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was diluted with 3 mL H₂O, anddialyzed in 12,000–14,000 MW cutoff tubing against water (2×2 L) for 48h. The solution was then removed from dialysis tubing and dried bylyophilization to yield 2.7 mg (30%) of 5,5′-dithiobis(2-nitrobenzoicacid)-1,4-bis(3-aminopropyl)piperazine-tris(2-(aminoethyl)aminecopolymer.

Example 33 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-Tetraethylenepentamine Copolymer

Tetraethylenepentamine (3.2 μL, 0.017 mmol, Aldrich Chemical Company)was taken up in 1.0 mL dichloromethane and HCl (1 mL, 1 M in Et₂O,Aldrich Chemical Company) was added Et₂O was added and the resulting HClsalt was collected by filtration. The salt was taken up in 1 mL DMF and5,5′-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg, 0.017 mmol) wasadded. The resulting solution was heated to 80° C. anddiisopropylethylamine (15 μL, 0.085 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000–14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 5.8 mg (62%) of5,5′-dithiobis(2-nitrobenzoic acid)-tetraethylenepentamine copolymer.

Example 34 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoic acid)-TetraethylenepentamineCopolymer Complexes

Complexes were prepared as follows:

-   Complex I: pDNA (pCI Luc, 200 μg) was added to 300μL DMSO then 2.5    mL Ringers was added.-   Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then    5,5′-Dithiobis(2-nitrobenzoic acid)-Tetraethylenepentamine Copolymer    (336 μg) was added followed by 2.5 mL Ringers.

2.5 mL tail vain injections of the complex were performed as previouslydescribed. Results reported are for liver expression, and are theaverage of two mice. Luciferase expression was determined as previouslyreported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi,G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse musclein vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used.

250 μL Injections

-   Complex I: 25,200,000-   Complex II: 21,000,000

Results indicate that pDNA (pCI Luc)/5,5′-Dithiobis(2-nitrobenzoicacid)-tetraethylenepentamine copolymer complexes are nearly equivalentto pCI Luc DNA itself in 2.5 mL injections. This indicates that the pDNAis being released from the complex and is accessible for transcription.

Example 35 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer

Tetraethylenepentamine (2.3 μL, 0.012 mmol, Aldrich Chemical Company)and tris(2-aminoethyl)amine (0.51 μL, 0.0034 mmol, Aldrich ChemicalCompany) were taken up in 0.5 mL methanol and HCl (1 mL, 1 M in Et₂O,Aldrich Chemical Company) was added. Et₂O was added and the resultingHCl salt was collected by filtration. The salt was taken up in 1 mL DMFand 5,5′-dithiobis[succinimidyl (2-nitrobenzoate)] (10 mg, 0.017 mmol)was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (15 μL, 0.085 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000–14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 6.9 mg (77%) of5,5′-dithiobis(2-nitrobenzoic acid)-tetraethylenepentamine-tris(2-aminoethyl)amine copolymer.

Example 36 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer Complexes

Complexes were prepared as follows:

-   Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then 2.5    mL Ringers was added.-   Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then    5,5′-Dithiobis(2-nitrobenzoic    acid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer (324    μg) was added followed by 2.5 mL Ringers.

2.5 mL tail vain injections of the complex were preformed as previouslydescribed. Results reported are for liver expression, and are theaverage of two mice. Luciferase expression was determined as previouslyreported (Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi,G., Jani, A. and Felgner, P. L. Direct gene transfer into mouse musclein vivo. Science, 1465–1468,1990.). A Lumat LB 9507 (EG&G Berthold,Bad-Wildbad, Germany) luminometer was used.

250 μL Injections

-   Complex 1: 25,200,000-   Complex II: 37,200,000

pDNA (pCI Luc)/5,5′-Dithiobis(2-nitrobenzoicacid)-tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer Complexesare more effective than pCI Luc DNA in 2.5 mL injections. Indicatingthat the pDNA is released from the complex and is accessible fortranscription.

Example 37 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer

N,N′-Bis(2-aminoethyl)-1,3-propanediamine (2.8 μL, 0.017 mmol, AldrichChemical Company) was taken up in 1.0 mL dichloromethane and HCl (1 mL,1 M in Et₂O, Aldrich Chemical Company) was added. Et₂O was added and theresulting HCl salt was collected by filtration. The salt was taken up in1 mL DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000–14,000 MW cutoff tubing against water (2×2L) for 24 hr. The solution was then removed from dialysis tubing anddried by lyophilization to yield 5.9 mg (66%) of5,5′-dithiobis(2-nitrobenzoicacid)-N,N′-bis(2-aminoethyl)-1,3-propanediamine Copolymer.

Example 38 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer Complexes

Complexes were prepared as follows:

-   Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then 2.5    mL Ringers was added.-   Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then    5,5′-Dithiobis(2-nitrobenzoic    acid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer (474 μg)    was added followed by 2.5 mL Ringers.

Tail vain injections of 2.5 mL of the complex were preformed aspreviously described. Results reported are for liver expression, and arethe average of two mice. Luciferase expression was determined aspreviously reported.

Results: 2.5 mL Injections

-   Complex I: 25,200,000-   Complex II: 341,000

pDNA (pCI Luc)/5,5′-Dithiobis(2-nitrobenzoicacid)-tetraethylenepentamine Copolymer Complexes provides luciferaseexpression indicating that the pDNA is being released from the complexand is accessible for transcription.

Example 39 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amineCopolymer

N,N′-Bis(2-aminoethyl)-1,3-propanediamine (2.0 μL, 0.012 mmol, AldrichChemical Company) and tris(2-aminoethyl)amine (0.51 μL, 0.0034 mmol,Aldrich Chemical Company) were taken up in 0.5 mL methanol and HCl (1mL, 1 M in Et₂O, Aldrich Chemical Company) was added. Et₂O was added andthe resulting HCl salt was collected by filtration. The salt was takenup in 1 mL DMF and 5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg,0.017 mmol) was added. The resulting solution was heated to 80° C. anddiisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000–14,000 MW cutoff tubing against water (2×2L) for 24 hr. The solution was then removed from dialysis tubing anddried by lyophilization to yield 6.0 mg (70%) of5,5′-dithiobis(2-nitrobenzoicacid)-N,N′-bis(2-aminoethyl)-1,3-propanediamine-tris(2-aminoethyl)aminecopolymer.

Example 40 Mouse Tail Vein Injections of pDNA (pCILuc)/5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amineCopolymer Complexes

Complexes were prepared as follows:

-   Complex I: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then 2.5    mL Ringers was added.-   Complex II: pDNA (pCI Luc, 200 μg) was added to 300 μL DMSO then    5,5′-Dithiobis(2-nitrobenzoic    acid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amine    Copolymer (474 μg) was added followed by 2.5 mL Ringers.

Tail vain injections of 2.5 mL of the complex were preformed aspreviously described. Results reported are for liver expression, and arethe average of two mice. Luciferase expression was determined aspreviously reported.

Results: 2.5 mL Injections

-   Complex I: 25,200,000-   Complex II: 1,440,000

Results indicate that pDNA (pCI Luc)/5,5′-Dithiobis(2-nitrobenzoicacid)-N,N′-Bis(2-aminoethyl)-1,3-propaneddiamine-Tris(2-aminoethyl)amine Copolymer Complexes are less effective than pCILuc DNA in 2.5 mL injections. Although the complex was less effective,the luciferase expression indicates that the pDNA is being released fromthe complex and is accessible for transcription.

Example 41 Intramuscular Injections of Complexes from pDNA (pCILuc)/Physiologically Labile Disulfide Bond Containing Polymers on Mouse.

Seven complexes were prepared as follows:

-   Complex I: pDNA (pCI Luc, 40 μg) was added to 586 μL glucose (290    mM)-HEPES (5 mM, pH 8).-   Complex II: pDNA (pCI Luc, 40 μg) was added to 577 μL glucose (290    mM)-HEPES (5 mM, pH 8). To this solution was added    5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)piperazine    Copolymer (9 μL, 200 μg).-   Complex III: pDNA (pCI Luc, 40 μg) was added to 573 μL glucose (290    mM)-HEPES (5 mM, pH 8). To this solution was added    5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)piperazine    Copolymer (13 μL, 200 μg).-   Complex IV: pDNA (pCI Luc, 40 μg) was added to 574 μL glucose (290    mM)-HEPES (5 mM, pH 8). To this solution was added    5,5′-Dithiobis(2-nitrobenzoic acid)-Tetraethylenepentamine Copolymer    (12 μL, 70 μg).-   Complex V: pDNA (pCI Luc, 40 μg) was added to 576 μL glucose (290    mM)-HEPES (5 mM, pH 8). To this solution was added    5,5′-Dithiobis(2-nitrobenzoic    acid)-Tetraethylenepentamine-Tris(2-aminoethyl)amine Copolymer (10    μL, 65 μg).-   Complex VI: pDNA (pCI Luc, 40 μg) was added to 581 μL glucose (290    mM)-HEPES (5 mM, pH 8). To this solution was added    5,5′-Dithiobis(2-nitrobenzoic    acid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine Copolymer (5 μL, 94    μg).-   Complex VII: pDNA (pCI Luc, 40 μg) was added to 570 μL glucose (290    mM)-HEPES (5 mM, pH 8). To this solution was added    5,5′-Dithiobis(2-nitrobenzoic    acid)-N,N′-Bis(2-aminoethyl)-1,3-propanediamine-Tris(2-aminoethyl)amine    Copolymer (16 μL, 94 μg).

Direct muscle injections of 150 μL of the complex were preformed aspreviously described (See Budker, V., Zhang, G., Danko, I., Williams,P., and Wolff, J., “The Efficient Expression Of IntravascularlyDelivered DNA In Rat Muscle,” Gene Therapy 5, 272–6(1998); Wolff, J. A.,Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. andFelgner, P. L. Direct gene transfer into mouse muscle in vivo. Science,1465–1468, 1990. which are incorporated herein by reference.). Sevendays post injection, the animals were sacrificed, and the muscleharvested. Samples were homogenized in lux buffer (1 mL), andcentrifuged for 15 minutes at 4000 RPM. Luciferase expression wasdetermined as previously reported. Results reported for left quadracep:right quadracep (Complex IV-only injected into left quadracep).

Results:

-   Complex I: RLU=1,900: 4,316-   Complex II: RLU=13,433: 20,640-   Complex III: RLU=10,156 : 39,491-   Complex IV: RLU=9,888:-   Complex V: RLU=19,565: 5,806-   Complex VI: RLU=270: 427-   Complex VII: RLU=973: 6,000

The complexes prepared from pCI Luc DNA/Physiologically Labile DisulfideBond Containing Polymers are effective in direct muscle injections. Theluciferase expression indicates that the pDNA is being released from thecomplex and is accessible for transcription. Complexes prepared with5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)piperazineCopolymer were the most effective, giving luciferase expression levels 2to 10 times as high as pDNA.

Example 42 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-Pentaethylenehexamine Copolymer

Pentaethylenehexamine (4.2 μL, 0.017 mmol, Aldrich Chemical Company) wastaken up in 1.0 mL dichloromethane and HCl (1 mL, 1 M in Et₂O, AldrichChemical Company) was added Et₂O was added and the resulting HCl saltwas collected by filtration. The salt was taken up in 1 mL DMF and5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017 mmol) wasadded. The resulting solution was heated to 80° C. anddiisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000–14,000 MW cutoff tubing against water (2×2L) for 24 hr. The solution was then removed from dialysis tubing anddried by lyophilization to yield 5.9 mg (58%) of5,5′-dithiobis(2-nitrobenzoic acid)-pentaethylenehexamine Copolymer.

Example 43 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-Pentaethylenehexamine-Tris(2-aminoethyl)amine Copolymer

Pentaethylenehexamine (2.9 μL, 0.012 mmol, Aldrich Chemical Company) andtris(2-aminoethyl)amine (0.51 μL, 0.0034 mmol, Aldrich Chemical Company)were taken up in 0.5 mL methanol and HCl (1 mL, 1 M in Et₂O, AldrichChemical Company) was added. Et₂O was added and the resulting HCl saltwas collected by filtration. The salt was taken up in 1 mL DMF and5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (10 mg, 0.017 mmol) wasadded. The resulting solution was heated to 80° C. anddisopropylethylamine (12 μL, 0.068 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000–14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 6.0 mg (64%) of5,5′-dithiobis(2-nitrobenzoicacid)-pentaethylenehexamine-tris(2-aminoethyl)amine copolymer.

Example 44 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-N-(3-Aminopropyl)-1,3-propanediamine Copolymer

5,5′-Dithiobis[succinimidyl(2-nitrobenzoate)] (2.5 mg, 0.0042 mmol) wastaken up in 10 μL of DMF. N-(3-aminopropyl)-1,3-propanediamine (0.6 μL,0.004 mmol, Aldrich Chemical Company) was added with 10 μL HEPES 250 mM,pH 7.5. After 1 hr the solution was concentrated under reduced pressure.The resulting residue was dissolved in 0.42 mL DMSO. Analysis of thesolution on SDS-PAGE versus poly-L-lysisne hydrobromide (MW of 1000,7500, 15000) indicated an approximate molecular weight range of3500–8000 for the polymer.

Example 45 Synthesis of 5,5′-dithiobis(2-nitrobenzoicacid)-1,4-bis(3-aminopropyl)piperazine-Folate Copolymer

Folate-PEG(3400 MW)-NH2 was prepared according to the known procedure(Lee, R. J., Low, P. S. Biochimica et Biophysica Acta 1233, 1995,134–144). Folate-PEG-NH2 was acylated with succinylatedN-(3-(BOC)aminopropyl)-1,3-propaneamine(BOC)amine. Removal of the BOCprotecting groups afforded the Folate monomer.1,4-bis(3-aminopropyl)piperazine (5.0 μL, 0.023 mmol, Aldrich ChemicalCompany) and folate monomer (5.0 mg, 0.0012 mmol) were taken up in 0.4mL methanol and HCl (1 mL, 1 M in Et₂O, Aldrich Chemical Company) wasadded. The resulting suspension was concentrated under reduced pressureto afford a white solid. The salt was taken up in 0.5 mL DMF and5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (14 mg, 0.025 mmol) wasadded. The resulting solution was heated to 80° C. anddiisopropylethylamine (18 μL, 0.10 mmol, Aldrich Chemical Company) wasadded by drops. After 16 hr, the solution was cooled, diluted with 3 mLH₂O, and dialyzed in 12,000–14,000 MW cutoff tubing against water (2×2L) for 24 h. The solution was then removed from dialysis tubing anddried by lyophilization to yield 13 mg (68%) of5,5′-dithiobis(2-nitrobenzoic acid)-1,4-bis(3-aminopropyl)piperazine-folate copolymer.

Example 46 Synthesis of 5,5′-Dithiobis(2-nitrobenzoicacid)-Poly-Glutamicacid (8mer) Copolymer

H₂N-EEEEEEEE-NHCH₂CH₂NH₂ (SEQ ID 5) (5.0 mg, 0.0052 mmol, Genosis) wastaken up in 0.1 mL HEPES (250 mM, pH 7.5).5,5′-dithiobis[succinimidyl(2-nitrobenzoate)] (3.1 mg, 0.0052) was addedwith 0.2 mL DMSO and the mixture was stirred overnight at roomtemperature. After 16 hr the solution was heated to 70° C. for 10 min,cooled to room temperature and diluted to 1.10 mL with DMSO.

Example 47 Complex Formation with 5,5′-Dithiobis(2-nitrobenzoicacid)-Poly-Glutamicacid (8mer) Copolymer

Fluorescein labeled DNA was used for the determination of DNAcondensation in complexes with 5,5′-Dithiobis(2-nitrobenzoic acid)Poly-Glutamicacid (8mer) Copolymer. pDNA was modified to a level of 1fluorescein per 20 bases using Minis' LabelIT™ Fluorescein kit. Thefluorescence was determined using a fluorescence spectrophotometer(Shimadzo RF-1501 Fluorescence Spectrophotometer), at an excitationwavelength of 497 nm, and an emission wavelength of 520 nm.

To fluorescein labeled DNA (10 μg) in 1 mL HEPES (25 mM, pH 7.5) wasadded polyornithine (18 μg, Sigma Chemical Company). The mixtures wereheld at room temperature for 5 minutes and the fluorescence wasdetermined. (see: Trubetskoy, V. S., Slattum, P. M., Hagstrom, J. E.,Wolff, J. A., Budker, V. G., “Quantitative Assessment of DNACondensation,” Anal. Biochem (1999) incorporated by reference) Sincefluorescence intensity is decreased by DNA condensation, resultsindicate that polyornithine compacts DNA. To the resulting complex wasadded 5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer)Copolymer (60 μg), and the fluorescence was again determined. Thefluorescence of the sample decreased further.

Upon the addition of 5,5′-Dithiobis(2-nitrobenzoicacid)-Poly-Glutamicacid (8mer) Copolymer to the sample, the fluorescencedecreased, indicating the formation a triple complex. No competition ofthe 5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer)Copolymer for the polyornithine was observed (increase in fluorescence).

Example 48 Transfection of 3T3 Cells with 5,5′-Dithiobis(2-nitrobenzoicacid)-Poly-Glutamicacid (8mer) Copolymer

Three complexes were formed:

-   Complex I) To 300 μL Opti-MEM was added LT-1TM (12 μg, Mirus    Corporation) followed by pDNA (pCI Luc, 4 μg).-   Complex II) To 300 μL Opti-MEM was added LT-1™ (12 μg, Mirus    Corporation) followed by pDNA (pCI Luc, 4 μg), and    5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer)    Copolymer (4 μg).-   Complex III) To 300 μL Opti-MEM was added LT-1™ (12 μg, Mirus    Corporation) followed by pDNA (pCI Luc, 4 μg), and Poly-Glutamicacid    (4 μg, MW 49000, Sigma Chemical Company).

Transfections were carried out in 35 mm wells. At the time oftransfection, 3T3 cells, at approximately 50% confluency, were washedonce with PBS (phosphate buffered saline), and subsequently stored inserum-free media (2.0 mL, Opti-MEM, Gibco BRL). 150 μL of complex wasadded to each well. After a 3.25 h incubation period at 37° C., themedia containing the complexes was aspirated from the cells, andreplaced with complete growth media, DMEM with 10% fetal bovine serum(Sigma). After an additional incubation of 48 hours, the cells wereharvested and the lysate was assayed for luciferase expression aspreviously reported (Wolff, J. A., Malone, R. W., Williams, P., Chong,W., Acsadi, G., Jani, A. and Felgner, P. L. Direct gene transfer intomouse muscle in vivo. Science, 1465–1468, 1990.). A Lumat LB 9507 (EG&GBerthold, Bad-Wildbad, Germany) luminometer was used.

Results:

-   Complex I: RLU=17,000,000-   Complex II: RLU=14,000,000-   Complex III: RLU=26,000,000

The addition of Poly-Glutamicacid (4 μg, MW 49000, Sigma ChemicalCompany) in the transfection experiment improved the pDNA expression.The addition of 5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid(8mer) Copolymer (4 μg) while not improving the pDNA expression was notdetrimental to the expression.

Example 49 Demonstration of Reduction of by5,5′-dithiobis(2-nitrobenzoic acid)-Containing Copolymers by Glutathione

To a solution of 5,5′-Dithiobis(2-nitrobenzoicacid)-1,4-Bis(3-aminopropyl)piperazine Copolymer (100 μg) in 0.5 mLHEPES (25 mM, pH 8) was added glutathione (final concentration of 2 mM).The absorbance of the sample was measured at λ 412 (The cleaveddisulfide has an absorbance maximum at λ 412. See Hermanson, G. T.Bioconjugate Techniques, Academic Press, New York, N.Y., 1996, pp 88)versus time (Beckman DU-7 Spectrophotometer).

To a solution of 5,5′-dithiobis(2-nitrobenzoicacid)-tetraethylenepentamine copolymer (50 μg) in 0.5 mL HEPES (25 mM,pH 8) was added glutathione (final concentration of 2 mM). Theabsorbance of the sample was measured at λ 412 (The cleaved disulfidehas an absorbance maximum at λ 412. See Hermanson, G. T. BioconjugateTechniques, Academic Press, New York, N.Y., 1996, pp 88) versus time(Beckman DU-7 Spectrophotometer).

To a solution of 5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid(8mer) Copolymer (50 μg) in 0.5 mL HEPES (25 mM, pH 8) was addedglutathione (final concentration of 2 mM). The absorbance of the samplewas measured at λ 412 (The cleaved disulfide has an absorbance maximumat λ 412. See Hermanson, G. T. Bioconjugate Techniques, Academic Press,New York, N.Y., 1996, pp 88) versus time (Beckman DU-7Spectrophotometer).

Each sample showed a rapid increase in the absorbance at λ 412 upon theaddition of glutathione, indicating cleavage of the disulfide bond. Halflife values were estimated as:

-   5,5′-Dithiobis(2-nitrobenzoic acid)-1,4-Bis(3-aminopropyl)piperazine    Copolymer t_(½)=42 sec.-   5,5′-dithiobis(2-nitrobenzoic acid)-tetraethylenepentamine copolymer    t_(½)=75 sec.-   5,5′-Dithiobis(2-nitrobenzoic acid)-Poly-Glutamicacid (8mer)    Copolymer t_(½)=24 sec.

The experiment demonstrates rapid cleavage of the disulfide bond of5,5′-dithiobis(2-nitrobenzoic acid)-containing copolymers with thephysiological reducing agent glutathione.

Example 50 Analysis of Delivery to Cells by VP22 Peptide:

Grow HeLa cells on glass coverslips by incubating at 4° C. in Delbecco'sModified Eagle's Media (DMEM) supplemented with 50 μg VP22peptide-fluorophore chimera (pulse). At this temperature, endocytosis isbelieved to be completely inhibited. Incubate the cells for two hours at4° C. and then wash with DMEM to remove external VP22-fluorophore.Remove the media and then either process cells for fluorescencemicroscopy or incubate three more hours at 4° C. with DMEM with mediachanges every hour (chase). The cells that are chased are then processedfor fluorescence microscopy. Cells processed for fluorescence microscopyare washed 3× in phosphate-buffered saline (PBS), fixed in PBS+4%formaldehyde for 20 min, washed 3× in PBS, and coverslips are mounted onslides. The presence of fluorophore is detected by confocal microscopy(Zeiss LSM 510).

Example 51 Analysis of Delivery to Cells by ANTP Peptide:

Grow HeLa cells on glass coverslips by incubating at 4° C. in Delbecco'sModified Eagle's Media (DMEM) supplemented with 50 μg ANTPpeptide-fluorophore chimera (pulse). At this temperature, endocytosis isbelieved to be completely inhibited. Incubate the cells for two hours at4° C. and then wash with DMEM to remove external ANTP-fluorophore.Remove the media and then either process cells for fluorescencemicroscopy or incubate three more hours at 4° C. with DMEM with mediachanges every hour (chase). The cells that are chased are then processedfor fluorescence microscopy. Cells processed for fluorescence microscopyare washed 3× in phosphate-buffered saline (PBS), fixed in PBS +4%formaldehyde for 20 min, washed 3× in PBS, and coverslips are mounted onslides. The presence of fluorophore is detected by confocal microscopy(Zeiss LSM 510).

The foregoing is considered as illustrative only of the principles ofthe invention. Furthermore, since numerous modifications and changeswill readily occur to those skilled in the art, it is not desired tolimit the invention to the exact construction and operation shown anddescribed. Therefore, all suitable modifications and equivalents fallwithin the scope of the invention.

1. A compound for delivering a molecule from outside a mammalian cell toinside said mammalian cell comprising: said molecule covalently linkedto a transduction signal via an activated disulfide bond that is cleavedmore rapidly than oxidized glutathione wherein said transduction signaltransports said molecule across a membrane of said cell, wherein saiddisulfide bond is cleaved in said cell and wherein said molecule remainsin said cell after two hours.
 2. The compound of claim 1 wherein thetransduction signal consists of a peptide with sequence substantiallyidentical to SEQ ID
 1. 3. The compound of claim 1 wherein thetransduction signal consists of VP22.
 4. The compound of claim 1 whereinthe transduction signal consists of ANTP.
 5. The compound of claim 1wherein the transduction signal consists of a polymer containing acationic charge.
 6. The compound of claim 5 wherein the transductionsignal consists of a peptide containing cationic residues.
 7. Thecompound of claim 1 wherein said molecule is associated with a nucleicacid.